WO2009046451A1 - Bactérie recombinante capable de susciter une réponse immunitaire protectrice contre c. perfringens - Google Patents

Bactérie recombinante capable de susciter une réponse immunitaire protectrice contre c. perfringens Download PDF

Info

Publication number
WO2009046451A1
WO2009046451A1 PCT/US2008/078993 US2008078993W WO2009046451A1 WO 2009046451 A1 WO2009046451 A1 WO 2009046451A1 US 2008078993 W US2008078993 W US 2008078993W WO 2009046451 A1 WO2009046451 A1 WO 2009046451A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
bacterium
antigen
expression
perfringens
Prior art date
Application number
PCT/US2008/078993
Other languages
English (en)
Inventor
Roy Curtiss, Iii
Bereket Zekarias
Kenneth Roland
Original Assignee
The Arizona Board Of Regents For And On Behalf Of Arizona State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Arizona Board Of Regents For And On Behalf Of Arizona State University filed Critical The Arizona Board Of Regents For And On Behalf Of Arizona State University
Priority to US12/681,711 priority Critical patent/US9040059B2/en
Publication of WO2009046451A1 publication Critical patent/WO2009046451A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention encompasses a recombinant bacterium capable of eliciting an immune response against Clostridium perfringens in a host.
  • C. perfringens is a ubiquitous gram positive, spore-forming, anaerobic organism, found in many environments surrounding poultry production, or other agricultural activities associated with animal rearing, including soil, dust, feces, feed, litter, rodents, and the intestinal contents of asymptomatic animals.
  • the toxins produced by C. perfringens strains cause necrotic enteritis (NE) in severe cases and have the ability, at lower doses, to cause a subclinical necrotic enteritis with thickening of the intestinal mucosa and decreased length of microvili in the ileum.
  • NE necrotic enteritis
  • perfringens colonization is to reduce the absorptive surface in the intestinal tract with a consequent reduction in the ability of birds, and most likely other animals including humans, to benefit from nutrients in food, resulting in a reduced rate of growth.
  • C. perfringens induces cellulitis and gangrenous dermatitis and is becoming an increasing concern in turkeys as well. Additionally, it is a frequent cause of gas gangrene in humans.
  • C. perfringens can cause a range of health problems in infected birds, ranging from a subclinical infection which can result in poor feed conversion caused by decreased digestion and adsorption, to necrotic enteritis, resulting in a variety of symptoms including severe depression, decreased appetite, reluctance to move, diarrhea and ruffled feathers, often leading to death.
  • Clinical illness is usually short, with birds often simply found dead. Onset of disease symptoms generally occurs in broilers from two to five weeks of age, coinciding with the disappearance of maternal antibodies.
  • NE has also been reported in layers of various ages. Gross lesions typically involve the ileum and jejunum, although cecal lesions can occur.
  • Intestines are friable and distended with gas and fluid and a diphtheritic membrane is often found in the mucosa.
  • Subclinical infection with C. perfringens can lead to economic losses, due to reduced growth rates and poor feed conversion. It is likely that losses due to subclinical infections may constitute a larger problem overall than losses due to acute disease. Occasionally, cholagniohepatitis can result, leading to condemnation losses at slaughter.
  • One aspect of the invention encompasses a recombinant
  • Salmonella bacterium The bacterium is capable of the expression of at least one nucleic acid encoding at least one Clostridium perfringens antigen.
  • the bacterium when administered to a host, typically elicits an immune response against Clostridium perfringens.
  • the vaccine composition comprises a recombinant bacterium capable of the expression of at least one nucleic acid encoding at least one Clostridium perfringens antigen.
  • the vaccine composition when administered to a host, when administered to a host, typically elicits an immune response against Clostridium perfringens.
  • Yet another aspect of the invention is a method of inducing an immune response against Clostridium perfringens.
  • the method comprises administering a composition comprising a recombinant bacterium capable of the expression of at least one nucleic acid encoding at least one Clostridium perfringens antigen to a host.
  • Fig. 1 depicts the expression of the PIcC antigen by the S. entehca serovar Typhimurium vaccine strain harboring different PIcC expression plasmids.
  • A Coomassie blue-stained SDS-PAGE. Lanes: M, molecular mass marker (in kilodaltons) (Bio-Rad, Hercules, CA); C, cytoplasmic fraction; P, pehplasmic fraction.
  • B Western blot of an analogous gel with rabbit anti-PlcC hyperimmune serum. Arrows show PIcC protein bands that also correspond to bands detected by western blotting.
  • FIG. 2 depicts PIcC vaccine antigen expression plasmids.
  • ss signal peptide sequence
  • CT C-terminal sequence
  • pYA4110 P ⁇ pp ompA ss vector The sequences of P frc , P/ pp , bla ss, ompA ss, and bla CT.
  • RBS ribosome binding site (Shine-Dalgarno sequence). Arrows show predicted signal peptide cleavage sites.
  • Fig. 3 depicts the predicted amino acid sequence and alignment of alpha toxin (PLC) mature protein sequences of strain CP995, an NE isolate, and ATCC 13124. Residues in CP995 that are different from those of ATCC 13124 are indicated.
  • FIG. 4 depicts ELISA results of anti-PicC serum IgG (A) and bile
  • Fig. 5 depicts an immunoblot of C. perfringens culture supernatant and rPlcC with ⁇ 8914(pYA3977)-immunized chicken serum.
  • Sera collected at 2 weeks after the boost immunization were pooled and used at a 1 :40 dilution.
  • C. perfringens culture supernatant was concentrated in 10% TCA (denaturing condition) or concentrated by filtration to retain the native structure.
  • Protein bands at about 43 kDa (closed arrow) in lanes 2 and 4 show the reactivity of serum antibody with alpha toxin, and lanes 5 and 6 show the reactivity with His-tagged rPlcC (19 kDa).
  • Lanes 1 and 3 C. perfringens culture supernatant (without concentration); lane 2, C. perfringens culture supernatant concentrated by 10% TCA precipitation; lane 4, C. perfringens culture supernatant concentrated by the Centhcon filtration system; lanes 5 and 6, purified rPlcC protein (open arrow).
  • FIG. 6 depicts DTH in oral RASV- or s.c. rPlcC-immunized chickens.
  • chickens Four weeks after immunization, chickens were injected with 20 ⁇ g purified rPlcC protein in the right leg toe web and with saline in the left leg toe web, and the swellings were measured 48 h later. The difference (diff.) in thickness between the left and right toe webs was calculated. Values are averages from five chickens per group.
  • Fig. 7 depicts a graph showing the neutralization of alpha toxin hemolytic activity by serum antibody.
  • the test serum final dilution was 1 :20.
  • Alpha toxin was obtained by concentrating the culture supernatant of an overnight culture of C. perfringens. The bars represent averages from five chickens per group.
  • FIG. 8 depicts indirect immunofluorescence detection of serum antibody binding to the bacterium surface.
  • C. perfringens smears were stained with nonimmunized control chicken serum (A), rPlcC protein injection immunized-chicken serum (B), and RASV-1 -primed and rPlcC parenteral boost-immunized chicken serum (C).
  • A nonimmunized control chicken serum
  • B rPlcC protein injection immunized-chicken serum
  • C RASV-1 -primed and rPlcC parenteral boost-immunized chicken serum
  • Fig. 9 depicts micrographs of gross lesions and lesion histopathology of challenged chickens.
  • A Gross lesions with hemorrhagic spots in the jejunum in control RAS-immunized chicken.
  • Jejunum distal region showing normal elongated villi.
  • C Degeneration and sloughing of apical villus epithelium.
  • D Severe necrotic lesion on villi and inflammatory cell infiltration (i.e., widened lamina intestinal).
  • E Higher magnification of villus tip showing clumps of bacteria (arrow) in gut lumen and attached to villus tips, often without a major lesion on the surface epithelium in immunized chickens.
  • Fig. 10 depicts the optimized plcC sequence.
  • FIG. 11 depicts diagrams of vectors pYA4531 (A) and pYA4538 (B).
  • Fig. 12 depicts a western blot showing PIcC synthesis from codon- optimized plcC in recombinant avirulent Salmonella.
  • Fig. 13 depicts the optimized netB sequence.
  • Fig. 14 depicts diagrams of vectors pYA4676 (A) and pYA4677 (B).
  • Fig. 15 depicts a western blot showing NetB synthesis from codon- optimized plcC in recombinant avirulent Salmonella.
  • Fig. 16 depicts the optimized tPFOR sequence.
  • Fig. 17 depicts the optimized tHP sequence.
  • Fig. 18 depicts the optimized GDP sequence.
  • Fig. 19 depicts the optimized FBA sequence.
  • Fig. 20 depicts a diagram of vector pYA4555.
  • Fig. 21 depicts a diagram of vector pYA4555 + bla SS netB (optimized).
  • Fig. 22 depicts a diagram of vector pYA4679.
  • the present invention encompasses a recombinant bacterium capable of eliciting an immune response against C. perfringens.
  • a vaccine composition comprising a recombinant bacterium of the invention may provide economic benefit to the poultry producer by inducing an immune response to C. perfringens. The immune response would result in enhanced feed conversion efficiency and more rapid growth, without the costs or potential human health hazards of using growth-promoting antibiotics. Additionally, the vaccine composition may substantially reduce Salmonella infection of poultry and thus contamination of carcasses and eggs. Therefore, use of the vaccine composition of the invention may contribute to reducing the likelihood of human infection due to Salmonella transmission through the food chain.
  • One aspect of the present invention encompasses a recombinant
  • Salmonella bacterium when administered to a host, typically elicits an immune response against C. perfringens.
  • the bacterium is capable of the expression of at least one nucleic acid encoding at least one C. perfringens antigen.
  • the recombinant bacterium is also capable of the regulated expression of a nucleic acid encoding at least one serotype-specific antigen of the bacterium.
  • the bacterium does not substantially induce an immune response specific to the serotype of the recombinant bacterium.
  • a recombinant Salmonella bacterium of the invention is capable of colonizing a host to substantially the same extent as a wild- type bacterium of the same serotype.
  • a bacterium of the invention will preferably be substantially avirulent after colonization.
  • the recombinant bacterium may be a
  • a bacterium of the invention may be derived from S. Typhimurium, S. Typhi, S. Paratyphi, S. Gallinarum, S. Enteritidis, S. Choleraesius, S. Ahzonae, or S. Dublin.
  • a bacterium of the invention may be derived from S. Typhimurium, S. Enteriditis, or S. Gallinarum.
  • a recombinant bacterium of the invention generally does not comprise any drug resistance nucleic acid sequences or other sequence scars in the chromosomes of the recombinant strain.
  • a recombinant bacterium of the invention may be capable of the regulated expression of a nucleic acid encoding at least one serotype-specific antigen.
  • serotype-specific antigen refers to an antigen that elicits an immune response specific for the bacterial vector serotype.
  • the immune response to a serotype-specific antigen may also recognize closely related strains in the same serogroup, but in a different, but related, serotype.
  • Non-limiting examples of serotype-specific antigens may include LPS O- antigen, one or more components of a flagellum, and Vi capsular antigen.
  • the expression of at least one, at least two, at least three, or at least four nucleic acid sequences encoding a serotype-specific antigen may be regulated in a bacterium of the invention.
  • the phrase "regulated expression of a nucleic acid encoding at least one serotype-specific antigen” refers to expression of the nucleic acid encoding a serotype-antigen such that the bacterium does not substantially induce an immune response specific to the bacterial vector serotype.
  • the expression of the serotype-specific antigen is eliminated.
  • the expression is substantially reduced.
  • the expression of the serotype- specific antigen is reduced in a temporally controlled manner. For instance, the expression of the serotype-specific antigen may be reduced during growth of the bacterium in a host, but not during in vitro growth.
  • nucleic acid encoding a Salmonella serotype-specific antigen may be measured using standard molecular biology and protein chemistry techniques known to one of skill in the art.
  • substantially reduction of the expression of a nucleic acid encoding a serotype-specific antigen refers to a reduction of at least about 1 % to at least about 99.9% as compared to a Salmonella bacterium in which no attempts have been made to reduce serotype-specific antigen expression.
  • the expression of a nucleic acid encoding a serotype-specific antigen is reduced by 100% by using a deletion mutation.
  • the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 % or 90%. In yet other embodiments of the invention, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81 % or 80%.
  • the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 75%, 70%, 65%, 60%, 55%, or 50%. In additional embodiments, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 45%, 40%, 35%, 30%, 25%, or 20%. In yet additional embodiments, the expression of a nucleic acid encoding a serotype-specific antigen is reduced by at least about 15%, 10%, 5%, 4%,3%, 2% or 1 %.
  • the expression of a nucleic acid encoding the serotype-specific antigen LPS O-antigen is regulated by mutating the pmi nucleic acid sequence, which encodes a phosphomannose isomerase needed for the bacterium to interconvert fructose-6-P and mannose-6-P.
  • the bacterium comprises a ⁇ pmi mutation, such as a ⁇ pmi-2426 mutation.
  • a bacterium comprising a ⁇ pmi-2426 mutation, grown in the presence of mannose, is capable of synthesizing a complete LPS O-antigen. But non-phosphorylated mannose, which is the form required for bacterial uptake, is unavailable in vivo.
  • a bacterium comprising a ⁇ pmi-2426 mutation loses the ability to synthesize LPS O-antigen serotype specific side chains after a few generations of growth in vivo.
  • the LPS that is synthesized comprises a core structure that is substantially similar across many diverse Salmonella serotypes. This results in a bacterium that is capable of eliciting an immune response against at least two Salmonella serotypes without substantially inducing an immune response specific to the serotype of the bacterial vector.
  • a bacterium of the invention that comprises a Apmi mutation may also comprise other mutations that ensure that mannose available to the bacterium during in vitro growth is used for LPS O-antigen synthesis.
  • a bacterium may comprise a A(gmd-fcl)-26 mutation. This mutation deletes two nucleic acid sequences that encode enzymes for conversion of GDP-mannose to GDP-fucose. This ensures that mannose available to the bacterium during in vitro growth is used for LPS O-antigen synthesis and not colanic acid production.
  • a bacterium may comprise the ⁇ (wza-wcaM)-8 mutation, which deletes all 19 nucleic acid sequences necessary for colanic acid production, and also precludes conversion of GDP-mannose to GDP-fucose.
  • LPS O-antigen may be regulated by arabinose, which is also absent in vivo.
  • a bacterium may comprise the mutation ⁇ P,f C ;;TT araC P BAD rfc. (P stands for promoter and TT stands for transcription terminator.)
  • the rfc (otherwise known as wzy) nucleic acid sequence is necessary for the addition of O-antigen subunits, which typically comprise three or four sugars, in a repeat fashion. When the rfc nucleic acid sequence is absent, only one O-antigen repeat subunit is added to the LPS core polysaccharide.
  • the serotype-specific O-antigen contains some 50 or so repeats of the O-antigen subunit, catalyzed by the enzyme encoded by the rfc nucleic acid sequence.
  • expression of the rfc nucleic acid sequence is dependant on the presence of arabinose that can be supplied during in vitro growth of the strain, but that is absent in vivo. Consequently, rfc expression ceases in vivo, resulting in the cessation of assembly of the O-antigen repeat structure.
  • Another means to regulate LPS O-antigen expression is to eliminate the function of galE in a recombinant bacterium of the invention.
  • the galE nucleic acid sequence encodes an enzyme for the synthesis of UDP-GaI, which is a substrate for LPS O-antigen, the outer LPS core and colanic acid. Growth of a bacterium comprising a suitable galE mutation in the presence of galactose leads to the synthesis of O-antigen and the LPS core.
  • Non-phosphorylated galactose is unavailable in vivo, however, and in vivo synthesis of UDP-GaI ceases, as does synthesis of the O- antigen and the LPS outer core.
  • One example of a suitable galE mutation is the ⁇ (galE- ybhC)-851 mutation.
  • a bacterium of the invention may comprise one or more of the ⁇ pmi, ⁇ P rfc ::TT araC P BAD rfc, and ⁇ galE mutations, with or without a ⁇ (gmd-fcl)-26 or ⁇ (wza-wcaM)-8 mutation.
  • Such a combination may yield a recombinant bacterium that synthesizes all components of the LPS core and O-antigen side chains when grown in vitro (i.e.
  • a recombinant bacterium with the inability to synthesize the LPS outer core and/or O-antigen is attenuated, as the bacterium is more susceptible to macrophages and/or complement-mediated cytotoxicity.
  • a bacterium with the inability to synthesize the LPS outer core and O-antigen in vivo induces only a minimal immune response to the serotype-specific LPS O-antigen.
  • a serotype-specific component of a flagellum is regulated by mutating the nucleic acid that encodes FIjB or FIiC.
  • a bacterium of the invention may comprise a ⁇ fljB217 mutation.
  • a bacterium may comprise a ⁇ fliCI80 mutation.
  • the ⁇ fljB217 mutation deletes the structural nucleic acid sequence that encodes the Phase Il flagellar antigen whereas the ⁇ fliCI80 mutation deletes the 180 amino acids encoding the antigenically variable serotype-specific domain of the Phase I FIiC flagellar antigen.
  • the portion of the flagellar protein that interacts with TLR5 to recruit/stimulate innate immune responses represents the conserved N-and C-terminal regions of the flagellar proteins and this is retained and expressed by strains with the AfliCI80 mutation. In addition, the ⁇ fliCI80 mutation retains the CD4-dependent T-cell epitope. It should be noted, that expression of the Phase I flagellar antigen and not the Phase Il flagellar antigen potentiates S.Typhimurium infection of mice. S.
  • Typhimurium recombinant bacteria with the ⁇ pmi-2426, ⁇ fljB2l7 and ⁇ fliC180 mutations when grown in the absence of mannose, are not agglutinated with antisera specific for the B-group O-antigen or the S. Typhimurium specific anti-flagellar sera. These recombinant bacteria are also non- motile since the FIiCI 80 protein that is synthesized at high levels is not efficiently incorporated into flagella.
  • recombinant bacteria When these recombinant bacteria are evaluated using HEK293 cells specifically expressing TLR5, the level of NFKB production is about 50% higher than when using a ⁇ fliB217 F1 iC+ strain that assembles flagellin into flagella and exhibits motility (there is no NFKB production by the control ⁇ fljB217 ⁇ fliC2426 strain with no flagella).
  • recombinant bacteria with the ⁇ (galE- ybhC)-851, ⁇ fljB2l7 and ⁇ fliC180 mutations when grown in the absence of galactose, are not agglutinated with antisera specific for the B-group O-antigen or the S. Typhimurium specific anti- flagellar sera. These bacteria are also non-motile.
  • Certain Salmonella strains such as S. Typhi and S. Dublin, express the Vi capsular antigen.
  • This antigen is serotype-specific, inhibits invasion, and acts to suppress induction of a protective immune response. Consequently, when a recombinant bacterium of the invention is derived from a strain comprising the Vi capsular antigen, one or more nucleic acids encoding the Vi capsular antigen will be deleted such that the Vi capsular antigen is not synthesized.
  • a recombinant bacterium of the invention is capable of the expression of at least one nucleic acid encoding at least one C. perfringens antigen.
  • C. perfringens antigen refers to an antigen that elicits an immune response against C. perfringens.
  • the C. perfringens antigen elicits a protective immune response against C. perfringens.
  • Non-limiting examples of C. perfringens antigens may include PIcC, or NetB (otherwise referred to as CpbX), GDP, PFOR, FBA, HP, or a fragment thereof.
  • the antigen is NetB, tPFOR, or tHP.
  • nucleic acid encoding a C. perfringens antigen comprise the complete nucleic acid sequence of the antigen. It is only necessary that the C. perfringens antigen sequence used be capable of eliciting an immune response.
  • the antigen may be one that was not found in that exact form in the parent organism. For example, a sequence coding for an antigen comprising 100 amino acid residues may be transferred in part into a recombinant bacterium so that a peptide comprising only 75, 65, 55, 45, 35, 25, 15, or even 10, amino acid residues is produced by the recombinant bacterium. Alternatively, if the amino acid sequence of a particular C.
  • perfringens antigen or fragment thereof is known, it may be possible to chemically synthesize the nucleic acid fragment or analog thereof by means of automated nucleic acid sequence synthesizers, PCR, or the like and introduce said nucleic acid sequence into the appropriate copy number vector.
  • a C. perfringens antigen of the invention may comprise a B cell epitope or a T cell epitope.
  • an antigen to which an immune response is desired may be expressed as a fusion to a carrier protein that contains a strong promiscuous T cell epitope and/or serves as an adjuvant and/or facilitates presentation of the antigen to enhance, in all cases, the immune response to the antigen or its component part. This can be accomplished by methods known in the art. Fusion to tetnus toxin fragment C, CT-B, LT-B and hepatitis virus B core are particularly useful for these purposes, although other epitope presentation systems are well known in the art.
  • a nucleic acid sequence encoding an antigen of the invention may comprise a secretion signal.
  • suitable secretion signals may include the bla, dsbA, eltW-B, or ompA secretion signals.
  • an antigen of the invention may be toxic to the recombinant bacterium.
  • a recombinant bacterium may comprise a long sequence of nucleic acid encoding several nucleic acid sequence products, one or all of which may be C. perfringens antigens.
  • the expression of at least one, at least two, at least three, at least four, at least five, at least six, or more nucleic acids encoding C. perfringens antigens is regulated in a bacterium of the invention.
  • These antigens may be encoded by two or more open reading frames operably linked to be expressed coordinately as an operon, wherein each antigen is synthesized independently.
  • the two or more antigens may be encoded by a single open reading frame such that the antigens are synthesized as a fusion protein.
  • vectors as detailed below, may be used to express an antigen. For more details, see the examples.
  • the high level expression of a nucleic acid sequence encoding an antigen in a bacterium reduces the bacterium's fitness, such that the bacterium grows slowly, is susceptible to stresses encountered in the host, and is generally less able to effectively colonize effector lymphoid tissues.
  • High level expression of a nucleic acid sequence encoding an antigen is highly desirable to maximize induction of an immune response against the antigen. Consequently, the phrase "regulated expression of a nucleic acid encoding at least one C. perfringens antigen" refers to expression of the nucleic acid encoding at least one C.
  • perfringens antigen in a bacterium such that the bacterium is capable of colonizing a host at levels similar to a wild-type bacterium, and yet is still capable of eliciting an immune response against C. perfringens when administered to the host.
  • Methods of regulating expression of at least one C. perfringens antigen are discussed in detail below.
  • the expression of a nucleic acid sequence encoding a C. perfringens antigen is regulated by a chromosomally integrated nucleic acid sequence encoding a repressor and a vector.
  • a recombinant bacterium of the invention that is capable of the regulated expression of a nucleic acid sequence encoding at least one C. perfringens antigen may comprise, in part, at least one chromosomally integrated nucleic acid sequence encoding a repressor.
  • the nucleic acid sequence encoding a repressor is operably linked to a regulatable promoter.
  • the nucleic acid sequence encoding a repressor and/or the promoter may be modified from the wild-type nucleic acid sequence so as to optimize the expression level of the nucleic acid sequence encoding the repressor.
  • nucleic acid sequence encoding a repressor should not be integrated into a locus that disrupts colonization of the host by the recombinant bacterium, or attenuates the bacterium.
  • nucleic acid sequence encoding a repressor may be integrated into the relA nucleic acid sequence.
  • nucleic acid sequence encoding a repressor may be integrated into the endA nucleic acid sequence.
  • At least one nucleic acid sequence encoding a repressor is chromosomally integrated. In other embodiments, at least two, or at least three nucleic acid sequences encoding repressors may be chromosomally integrated into the recombinant bacterium. If there is more than one nucleic acid sequence encoding a repressor, each nucleic acid sequence encoding a repressor may be operably linked to a regulatable promoter, such that each promoter is regulated by the same compound or condition. Alternatively, each nucleic acid sequence encoding a repressor may be operably linked to a regulatable promoter, each of which is regulated by a different compound or condition.
  • repressor refers to a biomolecule that represses transcription from one or more promoters.
  • a suitable repressor of the invention is synthesized in high enough quantities during the in vitro growth of the bacterial strain to repress the transcription of the nucleic acid encoding an antigen of interest on the vector, as detailed below, and not impede the in vitro growth of the strain.
  • a suitable repressor will generally be substantially stable, i.e. not subject to proteolytic breakdown.
  • a suitable repressor will be diluted by about half at every cell division after expression of the repressor ceases, such as in a non-permissive environment (e.g. an animal or human host).
  • a repressor depends, in part, on the species of the recombinant bacterium used.
  • the repressor is usually not derived from the same species of bacteria as the recombinant bacterium.
  • the repressor may be derived from E. coli if the recombinant bacterium is from the genus Salmonella.
  • the repressor may be from a bacteriophage.
  • Suitable repressors are known in the art, and may include, for instance, Lacl of E. coli, C2 encoded by bacteriophage P22, or C1 encoded by bacteriophage ⁇ .
  • Other suitable repressors may be repressors known to regulate the expression of a regulatable nucleic acid sequence, such as nucleic acid sequences involved in the uptake and utilization of sugars.
  • the repressor is Lacl.
  • the repressor is C2.
  • the repressor is C1.
  • the chromosomally integrated nucleic acid sequence encoding a repressor is operably linked to a regulatable promoter.
  • promoter may mean a synthetic or naturally-derived molecule that is capable of conferring, activating or enhancing expression of a nucleic acid.
  • a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid.
  • operably linked means that expression of a nucleic acid is under the control of a promoter with which it is spatially connected.
  • a promoter may be positioned 5' (upstream) of the nucleic acid under its control.
  • the distance between the promoter and a nucleic acid to be expressed may be approximately the same as the distance between that promoter and the native nucleic acid sequence it controls. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
  • the regulated promoter used herein generally allows transcription of the nucleic acid sequence encoding a repressor while in a permissive environment (i.e. in vitro growth), but ceases transcription of the nucleic acid sequence encoding a repressor while in a non-permissive environment (i.e. during growth of the bacterium in an animal or human host). For instance, the promoter may be sensitive to a physical or chemical difference between the permissive and non-permissive environment. Suitable examples of such regulatable promoters are known in the art.
  • the promoter may be responsive to the level of arabinose in the environment.
  • arabinose may be present during the in vitro growth of a bacterium, while typically absent from host tissue.
  • the promoter is derived from an araC-P B AD system.
  • the araC-P B AD system is a tightly regulated expression system that has been shown to work as a strong promoter induced by the addition of low levels of arabinose.
  • the araC-araBAD promoter is a bidirectional promoter controlling expression of the araBAD nucleic acid sequences in one direction, and the araC nucleic acid sequence in the other direction.
  • the portion of the araC-araBAD promoter that mediates expression of the araBAD nucleic acid sequences, and which is controlled by the araC nucleic acid sequence product, is referred to herein as P BAD -
  • a cassette with the araC nucleic acid sequence and the araC-araBAD promoter may be used.
  • This cassette is referred to herein as araC-PBAD-
  • the AraC protein is both a positive and negative regulator of P BAD - In the presence of arabinose, the AraC protein is a positive regulatory element that allows expression from P BAD - In the absence of arabinose, the AraC protein represses expression from P BAD - This can lead to a 1 , 200-fold difference in the level of expression from P BAD - Full induction of P BAD transcription also requires binding of the Crp-cAMP complex, which is a key regulator of catabolite repression.
  • enteric bacteria contain arabinose regulatory systems homologous to the araC araBAD system from E. coli. For example, there is homology at the amino acid sequence level between the E. coli and the S. Typhimurium AraC proteins, and less homology at the DNA level. However, there is high specificity in the activity of the AraC proteins. For example, the E. coli AraC protein activates only E. coli P BAD (in the presence of arabinose) and not S.
  • an arabinose regulated promoter may be used in a recombinant bacterium that possesses a similar arabinose operon, without substantial interference between the two, if the promoter and the operon are derived from two different species of bacteria.
  • the concentration of arabinose necessary to induce expression is typically less than about 2%. In some embodiments, the concentration is less than about 1.5%, 1 %, 0.5%, 0.2%, 0.1 %, or 0.05%. In other embodiments, the concentration is 0.05% or below, e.g. about 0.04%, 0.03%, 0.02%, or 0.01 %. In an exemplary embodiment, the concentration is about 0.05%.
  • the promoter may be responsive to the level of maltose in the environment.
  • maltose may be present during the in vitro growth of a bacterium, while typically absent from host tissue.
  • the malT nucleic acid encodes MaIT, a positive regulator of four maltose-responsive promoters (P PQ , P EFG , P KBM , and P s ).
  • P PQ a positive regulator of four maltose-responsive promoters
  • P PQ a positive regulator of four maltose-responsive promoters
  • P PQ a positive regulator of four maltose-responsive promoters
  • P EFG a positive regulator of four maltose-responsive promoters
  • P KBM a positive regulator of four maltose-responsive promoters
  • P s maltose-responsive promoters
  • the combination of malT and a mal promoter creates a tightly regulated expression system that has been shown to work as a strong promoter induced by the addition of malto
  • the malEFG-malKBM promoter is a bidirectional promoter controlling expression of the malKBM nucleic acid sequences in one direction, and the malEFG nucleic acid sequences in the other direction.
  • P KBM the portion of the malEFG-malKBM promoter that mediates expression of the malKBM nucleic acid sequence, and which is controlled by the malT nucleic acid sequence product
  • P EFG - Full induction of P KBM requires the presence of the MaIT binding sites of P EFG -
  • a cassette with the malT nucleic acid sequence and one of the mal promoters may be used. This cassette is referred to herein as malT-P ma ⁇ . In the presence of malT-P ma ⁇ .
  • the promoter may be sensitive to the level of rhamnose in the environment.
  • the rhaRS-PrhaB activator-promoter system is tightly regulated by rhamnose. Expression from the rhamnose promoter (P rha ) is induced to high levels by the addition of rhamnose, which is common in bacteria but rarely found in host tissues.
  • the nucleic acid sequences rhaBAD are organized in one operon that is controlled by the PrhaBAD promoter.
  • RhaS and RhaR This promoter is regulated by two activators, RhaS and RhaR, and the corresponding nucleic acid sequences belong to one transcription unit that is located in the opposite direction of the rhaBAD nucleic acid sequences. If L-rhamnose is available, RhaR binds to the PrhaRs promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the PrhaBAD and the P r ha ⁇ promoter and activates the transcription of the structural nucleic acid sequences. Full induction of rhaBAD transcription also requires binding of the Crp-cAMP complex, which is a key regulator of catabolite repression.
  • L-arabinose and L-rhamnose act directly as inducers for expression of regulons for their catabolism, important differences exist in regard to the regulatory mechanisms.
  • L-Arabinose acts as an inducer with the activator AraC in the positive control of the arabinose regulon.
  • the L-rhamnose regulon is subject to a regulatory cascade; it is therefore subject to even tighter control than the araC P BAD system.
  • L-Rhamnose acts as an inducer with the activator RhaR for synthesis of RhaS, which in turn acts as an activator in the positive control of the rhamnose regulon.
  • rhamnose may be used to interact with the RhaR protein and then the RhaS protein may activate transcription of a nucleic acid sequence operably-linked to the PrhaBAD promoter.
  • the promoter may be sensitive to the level of xylose in the environment.
  • the xy/R-P xy ⁇ A system is another well-established inducible activator-promoter system.
  • Xylose induces xylose-specific operons (xylE, xylFGHR, and xylAB) regulated by XyIR and the cyclic AMP-Crp system.
  • the XyIR protein serves as a positive regulator by binding to two distinct regions of the xyl nucleic acid sequence promoters.
  • the xylR- PxyiAB and/or xy/R-P xy iFGH regulatory systems may be used in the present invention.
  • xylR P xy iAB xylose interacting with the XyIR protein activates transcription of nucleic acid sequences operably-l inked to either of the two P xy ⁇ promoters.
  • nucleic acid sequences of the promoters detailed herein are known in the art, and methods of operably-linking them to a chromosomally integrated nucleic acid sequence encoding a repressor are known in the art and detailed in the examples.
  • a nucleic acid sequence encoding a repressor and regulatable promoter detailed above, for use in the present invention may be modified so as to optimize the expression level of the nucleic acid sequence encoding the repressor.
  • the optimal level of expression of the nucleic acid sequence encoding the repressor may be estimated, or may be determined by experimentation. Such a determination should take into consideration whether the repressor acts as a monomer, dimer, trimer, tetramer, or higher multiple, and should also take into consideration the copy number of the vector encoding the antigen of interest, as detailed below.
  • the level of expression is optimized so that the repressor is synthesized while in the permissive environment (i.e.
  • the level of expression may be optimized by modifying the nucleic acid sequence encoding the repressor and/or promoter.
  • modify refers to an alteration of the nucleic acid sequence of the repressor and/or promoter that results in a change in the level of transcription of the nucleic acid sequence encoding the repressor, or that results in a change in the level of synthesis of the repressor.
  • modify may refer to altering the start codon of the nucleic acid sequence encoding the repressor.
  • a GTG or TTG start codon as opposed to an ATG start codon, may decrease translation efficiency ten-fold.
  • modify may refer to altering the Shine- Dalgarno (SD) sequence of the nucleic acid sequence encoding the repressor.
  • SD sequence is a ribosomal binding site generally located 6-7 nucleotides upstream of the start codon.
  • the SD consensus sequence is AGGAGG, and variations of the consensus sequence may alter translation efficiency.
  • modify may refer to altering the distance between the SD sequence and the start codon.
  • modify may refer to altering the -35 sequence for RNA polymerase recognition.
  • modify may refer to altering the -10 sequence for RNA polymerase binding.
  • modify may refer to altering the number of nucleotides between the -35 and -10 sequences.
  • modify may refer to optimizing the codons of the nucleic acid sequence encoding the repressor to alter the level of translation of the mRNA encoding the repressor. For instance, non-A rich codons initially after the start codon of the nucleic acid sequence encoding the repressor may not maximize translation of the mRNA encoding the repressor. Similarly, the codons of the nucleic acid sequence encoding the repressor may be altered so as to mimic the codons from highly synthesized proteins of a particular organism.
  • modify may refer to altering the GC content of the nucleic acid sequence encoding the repressor to enhance the stability of expression in Salmonella.
  • more than one modification or type of modification may be performed to optimize the expression level of the nucleic acid sequence encoding the repressor. For instance, at least one, two, three, four, five, six, seven, eight, or nine modifications, or types of modifications, may be performed to optimize the expression level of the nucleic acid sequence encoding the repressor.
  • the nucleic acid sequence of Lacl and the promoter may be altered so as to increase the level of Lacl synthesis.
  • the start codon of the Lacl repressor may be altered from GTG to ATG.
  • the SD sequence may be altered from AGGG to AGGA.
  • the codons of lacl may be optimized according to the codon usage for highly synthesized proteins of Salmonella.
  • the start codon of lacl may be altered, the SD sequence may be altered, and the codons of lacl may be optimized.
  • the chromosomally integrated nucleic acid sequence encoding the repressor further comprises a transcription termination sequence.
  • a transcription termination sequence may be included to prevent inappropriate expression of nucleic acid sequences adjacent to the chromosomally integrated nucleic acid sequence encoding the repressor and regulatable promoter.
  • a recombinant bacterium of the invention that is capable of the regulated expression of at least one nucleic acid sequence encoding a C. perfringens antigen may also comprise, in part, a vector.
  • the vector comprises a nucleic acid sequence encoding at least one C. perfringens antigen operably linked to a promoter.
  • the promoter is regulated by the chromosomally encoded repressor, such that the expression of the nucleic acid sequence encoding an antigen is repressed during in vitro growth of the bacterium, but the bacterium is capable of high level synthesis of the antigen in an animal or human host.
  • vector refers to an autonomously replicating nucleic acid unit.
  • the present invention can be practiced with any known type of vector, including viral, cosmid, phasmid, and plasmid vectors.
  • the most preferred type of vector is a plasmid vector.
  • plasmids and other vectors may possess a wide array of promoters, multiple cloning sequences, transcription terminators, etc., and vectors may be selected so as to control the level of expression of the nucleic acid sequence encoding an antigen by controlling the relative copy number of the vector.
  • the vector might encode a surface localized adhesin as the antigen, or an antigen capable of stimulating T-cell immunity, it may be preferable to use a vector with a low copy number such as at least two, three, four, five, six, seven, eight, nine, or ten copies per bacterial cell.
  • a non-limiting example of a low copy number vector may be a vector comprising the pSC101 ori.
  • an intermediate copy number vector might be optimal for inducing desired immune responses.
  • an intermediate copy number vector may have at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 copies per bacterial cell.
  • a non-limiting example of an intermediate copy number vector may be a vector comprising the p15A ori.
  • a high copy number vector might be optimal for the induction of maximal antibody responses or mucosal immune responses.
  • a high copy number vector may have at least 31 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 copies per bacterial cell.
  • a high copy number vector may have at least 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 copies per bacterial cell.
  • Non-limiting examples of high copy number vectors may include a vector comprising the pBR ori or the pUC ori.
  • vector copy number may be increased by selecting for mutations that increase plasmid copy number. These mutations may occur in the bacterial chromosome but are more likely to occur in the plasmid vector.
  • vectors used herein do not comprise antibiotic resistance markers to select for maintenance of the vector.
  • a vector may comprise one or more than one nucleic acid sequences encoding a C. perfringens antigen, whether regulated or not, as detailed above.
  • the vector comprises a nucleic acid sequence encoding at least one C. perfringens antigen operably-linked to a promoter regulated by the repressor, encoded by a chromosomally integrated nucleic acid sequence.
  • a repressor dictates, in part, the selection of the promoter operably-linked to a nucleic acid sequence encoding an antigen of interest.
  • the promoter may be selected from the group consisting of Lacl responsive promoters, such as P trc , P ⁇ ac , P ⁇ 7iac and P tac - If the repressor is C2, then the promoter may be selected from the group consisting of C2 responsive promoters, such as P22 promoters P L and P R . If the repressor is C1 , then the promoter may be selected from the group consisting of C1 responsive promoters, such as ⁇ promoters P L and P R .
  • the promoter regulates expression of a nucleic acid sequence encoding the antigen, such that expression of the nucleic acid sequence encoding an antigen is repressed when the repressor is synthesized (i.e. during in vitro growth of the bacterium), but expression of the nucleic acid sequence encoding an antigen is high when the repressor is not synthesized (i.e. in an animal or human host).
  • the concentration of the repressor will decrease with every cell division after expression of the nucleic acid sequence encoding the repressor ceases. In some embodiments, the concentration of the repressor decreases enough to allow high level expression of the nucleic acid sequence encoding a C.
  • the concentration of the repressor decreases enough to allow high level expression of the nucleic acid sequence encoding a C. perfringens antigen after about 5 divisions of the bacterium in an animal or human host.
  • the promoter may comprise other regulatory elements.
  • the promoter may comprise lacO if the repressor is Lacl. This is the case with the lipoprotein promoter P
  • the repressor is a Lacl repressor and the promoter is P trc .
  • the expression of the nucleic acid sequence encoding the C. perfringens antigen should be repressed when the repressor is synthesized. For instance, if the repressor is synthesized during in vitro growth of the bacterium, expression of the nucleic acid sequence encoding the C. perfringens antigen should be repressed. Expression may be “repressed” or “partially repressed” when it is about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1 %, or even less than 1 % of the expression under non-repressed conditions. Thus although the level of expression under conditions of "complete repression" might be exceeding low, it is likely to be detectable using very sensitive methods since repression can never by absolute.
  • the expression of the nucleic acid sequence encoding the antigen should be high when the expression of the nucleic acid sequence encoding the repressor is repressed. For instance, if the nucleic acid sequence encoding the repressor is not expressed during growth of the recombinant bacterium in the host, the expression of the nucleic acid sequence encoding the antigen should be high.
  • "high level" expression refers to expression that is strong enough to elicit an immune response to the antigen. Consequently, the copy number correlating with high level expression can and will vary depending on the antigen and the type of immune response desired.
  • the invention also encompasses other means of regulating the expression of a nucleic acid sequence encoding at least one C. perfringens antigen in a recombinant bacterium.
  • the C. perfringens antigen of interest may be encoded on an extra-chromosomal vector. This can be used in the context of a balanced-lethal host-vector system.
  • the nucleotide sequence encoding the antigen of interest may be inserted into the chromosome but have its expression controlled by a regulatable system, e.g., Lacl or C2, as with the regulated gene encoding the antigen of interest on an extra-chromosomal vector (e.g., a plasmid).
  • a regulatable system e.g., Lacl or C2
  • a recombinant bacterium of the invention capable of regulated expression may also be attenuated.
  • "Attenuated” refers to the state of the bacterium wherein the bacterium has been weakened from its wild type fitness by some form of recombinant or physical manipulation. This includes altering the genotype of the bacterium to reduce its ability to cause disease. However, the bacterium's ability to colonize the gut (in the case of Salmonella) and induce immune responses is, preferably, not substantially compromised.
  • regulated attenuation allows the recombinant bacterium to express one or more nucleic acids encoding products important for the bacterium to withstand stresses encountered in the host after immunization. This allows efficient invasion and colonization of lymphoid tissues before the recombinant bacterium is regulated to display the attenuated phenotype.
  • a recombinant bacterium may be attenuated as described in section l(a)i above, i.e. regulating LPS O-antigen.
  • a recombinant bacterium may be attenuated as described in section (c)i below.
  • both regulated attenuation and regulated expression of an enteric antigen encoding sequence may be dependent upon an arabinose regulatable system. Consequently, the concentration of arabinose needed for optimal expression of the regulated enteric antigen encoding sequence may not be the same as the concentration for optimal expression of attenuation.
  • the concentration of arabinose for the optimization of both regulated attenuation and regulated expression of sequences encoding antigen will be substantially the same.
  • the promoter and/or the nucleic acid sequence encoding an attenuation protein may be modified to optimize the system.
  • Methods of modification are detailed above. Briefly, for example, the SD hbosome binding sequence may be altered, and/or the start codon may be altered from ATG to GTG for the nucleic acid sequences fur and phoPQ, so that the production levels of Fur and PhoPQ are optimal for both the regulated attenuation phenotype and the regulated expression when growing strains with a given concentration of arabinose.
  • nuceic acid sequences, in addition to fur and phoPQ may also be altered as described herein in combination with other well-known protocols.
  • these attenuating nucleic acid sequences may be regulated by other systems using well-established protocols known to one of skill in the art. For example, they may be regulated using with promoters dependent on addition of maltose, rhamnose, or xylose rather than arabinose.
  • Attenuation may be accomplished by altering (e.g., deleting) native nucleic acid sequences found in the wild type bacterium.
  • nucleic acid sequences which may be used for attenuation include: a pab nucleic acid sequence, a pur nucleic acid sequence, an am nucleic acid sequence, asd, a dap nucleic acid sequence, nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc, poxA, galU, mviA, sodC, recA, ssrA, sirA, inv, hilA, rpoE, flgM, tonB, sly
  • the above nucleic acid sequences may be placed under the control of a sugar regulated promoter wherein the sugar is present during in vitro growth of the recombinant bacterium, but substantially absent within an animal or human host.
  • the cessation in transcription of the nucleic acid sequences listed above would then result in attenuation and the inability of the recombinant bacterium to induce disease symptoms.
  • the bacterium may also be modified to create a balanced-lethal host-vector system, although other types of systems may also be used (e.g., creating complementation heterozygotes).
  • the bacterium may be modified by manipulating its ability to synthesize various essential constituents needed for synthesis of the rigid peptidoglycan layer of its cell wall.
  • the constituent is diaminopimelic acid (DAP).
  • DAP diaminopimelic acid
  • Various enzymes are involved in the eventual synthesis of DAP.
  • the bacterium is modified by using a ⁇ asc//A mutation to eliminate the bacterium's ability to produce ⁇ -aspartate semialdehyde dehydrogenase, an enzyme essential for the synthesis of DAP.
  • a ⁇ asc//A mutation to eliminate the bacterium's ability to produce ⁇ -aspartate semialdehyde dehydrogenase, an enzyme essential for the synthesis of DAP.
  • One of skill in the art can also use the teachings of U.S. Patent No. 6,872,547 for other types of mutations of nucleic acid sequences that result in the abolition of the synthesis of DAP.
  • These nucleic acid sequences may include, but are not limited to, dapA, dapB, dapC, dapD, dapE, dapF, and asd.
  • modifications that may be employed include modifications to a bacterium's ability to synthesize D-alanine or to synthesize D- glutamic acid (e.g., ⁇ murl mutations), which are both unique constituents of the peptidoglycan layer of the bacterial cell wall.
  • Yet another balanced-lethal host-vector system comprises modifying the bacterium such that the synthesis of an essential constituent of the rigid layer of the bacterial cell wall is dependent on a nutrient (e.g., arabinose) that can be supplied during the growth of the microorganism.
  • a bacterium may -comprise the ⁇ P murA ::TT araC P BAD murA deletion-insertion mutation. This type of mutation makes synthesis of muramic acid (another unique essential constituent of the peptidoglycan layer of the bacterial cell wall) dependent on the presence of arabinose that can be supplied during growth of the bacterium in vitro.
  • Recombinant bacteria with a ⁇ P murA ::TT araC P BAD murA deletion-insertion mutation grown in the presence of arabinose exhibit effective colonization of effector lymphoid tissues after oral vaccination prior to undergoing lysis due to the inability to synthesize muramic acid.
  • various embodiments may comprise the araC PBAD c2 cassette inserted into the asd nucleic acid sequence that encodes aspartate semialdehyde dehydrogenase. Since the araC nucleic acid sequence is transcribed in a direction that could lead to interference in the expression of adjacent nucleic acid sequences and adversely affect vaccine strain performance, a transcription termination (TT) sequence is generally inserted 3' to the araC nucleic acid sequence.
  • the chromosomal asd nucleic acid sequence is typically inactivated to enable use of plasmid vectors encoding the wild-type asd nucleic acid sequence in the balanced lethal host-vector system. This allows stable maintenance of plasmids in vivo in the absence of any drug resistance attributes that are not permissible in live bacterial vaccines.
  • the wild-type asd nucleic acid sequence may be encoded by the vector described above.
  • AasdA27:.TT araC PBAD c2 has an improved SD sequence and a codon optimized c2 nucleic acid sequence.
  • the C2 repressor synthesized in the presence of arabinose is used to repress nucleic acid sequence expression from P22 P R and P L promoters.
  • AasdA27::TT araC P BAD c2 has the 1104 base-pair asd nucleic acid sequence deleted (1 to 1104, but not including the TAG stop codon) and the 1989 base-pair fragment containing T4 iplll TT araC P BAD C2 inserted.
  • the c2 nucleic acid sequence in ⁇ asdA27: :TT araC P BAD C2 has a SD sequence that was optimized to TAAGGAGGT. It also has an improved P BAD promoter such that the -10 sequence is improved from TACTGT to TATAAT. Furthermore, it has a codon optimized c2 nucleic acid sequence, in which the second codon was modified from AAT to AAA.
  • the bacterium may be attenuated by regulating the murA nucleic acid sequence encoding the first enzyme in muramic acid synthesis and the asd nucleic acid sequence essential for DAP synthesis.
  • These embodiments may comprise the chromosomal deletion-insertion mutations AasdA19::TT araC P BAD C2 or DasdA27::TT araC P BAD C2 and ⁇ P murA7 ::TT araC P BAD murA or DP murA i2::TT araC P BAD murA or ⁇ P murA 25::TT araC P BAD murA.
  • the recombinant bacterium may comprise araBAD and araE mutations to preclude breakdown and leakage of internalized arabinose such that asd and murA nucleic acid sequence expression continues for a cell division or two after oral immunization into an environment that is devoid of external arabinose.
  • plasmid vector pYA3681 contains the murA nucleic acid sequence (with altered start codon sequences to decrease translation efficiency) under the control of an araC P BAD promoter.
  • the second nucleic acid sequence under the direction of this promoter is the asd nucleic acid sequence (with altered start codon sequences to decrease translation efficiency).
  • the P22 P R promoter is in the anti-sense direction of both the asd nucleic acid sequence and the murA nucleic acid sequence.
  • the P22 P R is repressed by the C2 repressor made during growth of the strain in media with arabinose (due to the ⁇ asdAI 9::TT araC P BAD C2 deletion-insertion).
  • C2 concentration decreases due to cell division in vivo to cause P R directed synthesis of anti-sense mRNA to further block translation of asd and murA mRNA.
  • the araC P BAD sequence is also not from E.
  • coli B/r as originally described but represents a sequence derived from E. coli K-12 strain ⁇ 289 with tighter control and less leakiness in the absence of arabinose.
  • transcription terminators flank all of the domains for controlled lysis, replication, and expression so that expression in one domain does not affect the activities of another domain.
  • the plasmid asd nucleic acid sequence does not replace the chromosomal asd mutation since they have a deleted sequence in common, consequently, the E. coli murA nucleic acid sequence was used in the plasmid instead of using the Salmonella murA nucleic acid sequence.
  • the recombinant bacterium of this embodiment is avirulent at oral doses in excess of 10 9 CFU to BALB/c mice.
  • this construction exhibits complete biological containment with no in vivo recombinant bacteria survivors detectable after 21 days and no recombinant bacteria survivors during or after excretion. This property enhances vaccine safety and minimizes the potential for vaccination of individuals not intended for vaccination.
  • the present invention also encompasses a recombinant bacterium capable of regulated attenuation.
  • the bacterium comprises a chromosomally integrated regulatable promoter.
  • the promoter replaces the native promoter of, and is operably linked to, at least one nucleic acid sequence encoding an attenuation protein, such that the absence of the function of the protein renders the bacterium attenuated.
  • the promoter is modified to optimize the regulated attenuation.
  • a recombinant bacterium of the invention may comprise a regulatable promoter chromosomally integrated so as to replace the native promoter of, and be operably linked to, at least one nucleic acid sequence encoding an attenuation protein, such that the absence of the function of the protein renders the bacterium attenuated, and the bacterium may comprise another method of attenuation detailed in section I above.
  • an attenuation protein is meant to be used in its broadest sense to encompass any protein the absence of which attenuates a bacterium.
  • an attenuation protein may be a protein that helps protect a bacterium from stresses encountered in the gastrointestinal tract or respiratory tract.
  • Non-limiting examples may be the RpoS, PhoPQ, OmpR, Fur, and Crp proteins.
  • the protein may be a necessary component of the cell wall of the bacterium, such as the protein encoded by murA.
  • the protein may be listed in Section / above.
  • the native promoter of at least one, two, three, four, five, or more than five attenuation proteins may be replaced by a regulatable promoter as described herein.
  • the promoter of one of the proteins selected from the group comprising RpoS, PhoPQ, OmpR, Fur, and Crp may be replaced.
  • the promoter of two, three, four or five of the proteins selected from the group comprising RpoS, PhoPQ, OmpR, Fur, and Crp may be replaced.
  • each promoter may be replaced with a regulatable promoter, such that the expression of each attenuation protein encoding sequence is regulated by the same compound or condition.
  • each promoter may be replaced with a different regulatable promoter, such that the expression of each attenuation protein encoding sequence is regulated by a different compound or condition such as by the sugars arabinose, maltose, rhamnose or xylose.
  • the regulatable promoter used herein generally allows transcription of the nucleic acid sequence encoding the attenuation protein while in a permissive environment (i.e. in vitro growth), but cease transcription of the nucleic acid sequence encoding an attenuation protein while in a non-permissive environment (i.e. during growth of the bacterium in an animal or human host).
  • the promoter may be responsive to a physical or chemical difference between the permissive and non- permissive environment. Suitable examples of such regulatable promoters are known in the art and detailed above.
  • the promoter may be responsive to the level of arabinose in the environment, as described above. In other embodiments, the promoter may be responsive to the level of maltose, rhamnose, or xylose in the environment, as described above.
  • the promoters detailed herein are known in the art, and methods of operably linking them to a nucleic acid sequence encoding an attenuation protein are known in the art.
  • a recombinant bacterium of the invention may comprise any of the following: ⁇ P fur ::TT araC P BAD fur, ⁇ P crp ::TT araC P BAD crp, ⁇ P pho p Q ::TT araC P BAD phoPQ, or a combination thereof.
  • Growth of such strains in the presence of arabinose leads to transcription of the fur, phoPQ, and/or crp nucleic acid sequences, but nucleic acid sequence expression ceases in a host because there is no free arabinose. Attenuation develops as the products of the fur, phoPQ, and/or the crp nucleic acid sequences are diluted at each cell division.
  • the concentration of arabinose necessary to induce expression is typically less than about 2%. In some embodiments, the concentration is less than about 1.5%, 1 %, 0.5%, 0.2%, 0.1 %, or 0.05%. In certain embodiments, the concentration may be about 0.04%, 0.03%, 0.02%, or 0.01 %. In an exemplary embodiment, the concentration is about 0.05%. Higher concentrations of arabinose or other sugars may lead to acid production during growth that may inhibit desirable cell densities. The inclusion of mutations such as AaraBAD or mutations that block the uptake and/or breakdown of maltose, rhamnose, or xylose, however, may prevent such acid production and enable use of higher sugar concentrations with no ill effects.
  • the onset of attenuation may be delayed by including additional mutations, such as ⁇ araBAD23, which prevents use of arabinose retained in the cell cytoplasm at the time of oral immunization, and/or ⁇ araE25 that enhances retention of arabinose.
  • additional mutations such as ⁇ araBAD23, which prevents use of arabinose retained in the cell cytoplasm at the time of oral immunization, and/or ⁇ araE25 that enhances retention of arabinose.
  • inclusion of these mutations may be beneficial in at least two ways: first, enabling higher culture densities, and second enabling a further delay in the display of the attenuated phenotype that may result in higher densities in effector lymphoid tissues to further enhance immunogenicity.
  • Attenuation of the recombinant bacterium may be optimized by modifying the nucleic acid sequence encoding an attenuation protein and/or promoter. Methods of modifying a promoter and/or a nucleic acid sequence encoding an attenuation protein are the same as those detailed above with respect to repressors in Section (b).
  • more than one modification may be performed to optimize the attenuation of the bacterium. For instance, at least one, two, three, four, five, six, seven, eight or nine modifications may be performed to optimize the attenuation of the bacterium.
  • the SD sequences and/or the start codons for the fur and/or the phoPQ virulence nucleic acid sequences may be altered so that the production levels of these nucleic acid products are optimal for regulated attenuation.
  • Figure 8 depicts ⁇ P fur77 ::TT araC P BAD fur, whose start codon is changed from ATG to GTG, and ⁇ P fur8 i ::TT araC P BAD fur, that has a weakened SD sequence as well as the start codon changed from ATG to GTG.
  • Figure 9 depicts ⁇ P phO pQi73::TT araC P BAD phoPQ, that has modifications to the start codon as well as the second codon, which was changed from ATG to GTG.
  • Figure 9 also depicts ⁇ P P hoPQi77::TT araC P BAD phoPQ, wherein the SD sequence has been changed to the weaker AAGG sequence, the start codon was modified, and the second codon was modified from ATG to GTG.
  • a recombinant bacterium of the invention is generally capable of eliciting an immune response against at least two Salmonella serotypes. This may be accomplished, for instance, by eliminating the serotype-specific LPS O-antigen side chains as discussed above. The remaining LPS core will elicit an immune response, inducing the production of antibodies against the LPS core. Since this LPS core is substantially identical in the several thousand Salmonella enterica serotypes, the antibodies potentially provide immunity against several diverse Salmonella enterica serotypes, such as Typhimurium, Heidelberg, Newport, Infantis, Dublin, Hadar, Kentucky, Thompson, Agona, Ohio, Virchow, Typhi, Enteritidis, and Munchen.
  • the elimination of the LPS O-antigen provides the host immune system with better access to the outer membrane proteins of the recombinant bacterium, thereby enhancing induction of immune responses against these outer membrane proteins.
  • the outer membrane proteins may be upregulated to further enhance host immune responses to these proteins.
  • Non-limiting examples of outer membrane proteins include proteins involved in iron and manganese uptake, as described below. Iron and manganese are essential nutrients for enteric pathogens and the induction of antibodies that inhibit iron and manganese uptake in effect starves the pathogens, conferring protective immunity on the host. Additionally, since these proteins are homologous among the enteric bacteria, such host immune responses provide immunity against multiple Salmonella enterica serotypes.
  • the elicited immune response may include, but is not limited to, an innate immune response, a humoral immune response and a cell-mediated immune response.
  • Th2-dependent mucosal and systemic antibody responses to the C. perfringens antigen(s) are observed. Immune responses may be measured by standard immunological assays known to one of skill in the art.
  • the immune response is protective.
  • a recombinant bacterium of the invention may also comprise a ⁇ P C ⁇ ::TT araC P BAD crp deletion-insertion mutation. Since the araC P BAD cassette is dependent both on the presence of arabinose and the binding of the catabolite repressor protein Crp, a ⁇ P crp ::TT araC P BAD crp deletion-insertion mutation may be included as an additional means to reduce expression of any nucleic acid sequence under the control of the P BAD promoter. This means that when the bacterium is grown in a non-permissive environment (i.e.
  • a recombinant bacterium of the invention may be modified so as to reduce fluid secretion in the host.
  • the bacterium may comprise the ⁇ sopB1925 mutation.
  • the bacterium may comprise the ⁇ msbB48 mutation. For more details, see the Examples.
  • a live recombinant bacterium may possess the potential to survive and multiply if excreted from a host. This leads to the possibility that individuals not electing to be immunized may be exposed to the recombinant bacterium. Consequently, in certain embodiments, a recombinant bacterium of the invention may comprise one or more mutations that decrease, if not preclude, the ability of Salmonella vaccines to persist in the Gl tract of animals.
  • a recombinant bacterium of the invention may comprise one or more of the A(gmd fcl)-26 or A(wcaL-wza)-7, AagfBAC811 or ⁇ P agm agfG)-4, ⁇ bcsABZC2118 or ⁇ bcsEFG2319 and ⁇ (yshA-yihW)-157 mutations that block synthesis of colanic acid, thin aggregative fimbriae (i.e., curli), cellulose and extracellular polysaccharide, respectively, all of which contribute to biofilm formation.
  • vaccine strains with the A(wcaM-wza)-8 mutation synthesize five to ten percent more protective antigen and induce similarly higher antibody titers to this antigen.
  • recombinant bacterium comprising a biological containment mutation are not adversely effected in their virulence or the ability to colonize mice.
  • the recombinant bacterium may comprise a method of regulated delayed lysis in vivo that prevents bacterial persistence in vivo and survival if excreted.
  • mutations include: ⁇ (gmd-fcl)-26 that precludes synthesis of colanic acid that can protect cells undergoing cell wall-less death from lysing completely and AagfBAC811 that blocks synthesis of thin aggregative fimbriae (curli) that are critical for biofilm formation to enable persistent colonization on bile stones in the gall bladder, AasdA27::TT araC P BAD C2 insertion-deletion mutation to impose a requirement for the peptidoglycan constituent DAP and ⁇ P murA i2::TTaraC P BAD murA insertion- deletion mutation as a conditional-lethal mutation blocking synthesis of the peptidoglycan constituent muramic acid.
  • a regulated delayed lysis plasmid vector such as pYA3681 ( Figure 2) that has an arabinose-dependent expression of asdA and murA genes.
  • a recombinant bacterium comprising such mutations grows normally in the presence of arabinose. In vivo, however, the bacterium ceases to express any nucleic acids encoding the AsdA and MurA enzymes, such that synthesis of the peptidoglycan cell wall layer ceases, ultimately resulting in the lysis of the bacterium.
  • This lysis may result in the release of a bolus of antigen specific for an enteric pathogen, thereby serving as a means to enhance induction of immunity against that enteric pathogen while conferring complete biological containment.
  • a recombinant bacterium of the invention may be particularly suited for use as a vaccine.
  • Infection of a host with a Salmonella strain typically leads to colonization of the gut-associated lymphoid tissue (GALT) or Peyer's patches, which leads to the induction of a generalized mucosal immune response to the recombinant bacterium.
  • GALT gut-associated lymphoid tissue
  • Further penetration of the bacterium into the mesenteric lymph nodes, liver and spleen may augment the induction of systemic and cellular immune responses directed against the bacterium.
  • GALT gut-associated lymphoid tissue
  • the use of recombinant Salmonella for oral immunization stimulates all three branches of the immune system, which is particularly important for immunizing against infectious disease agents that colonize on and/or invade through mucosal surfaces.
  • a recombinant bacterium of the invention may be administered to a host as a vaccine composition.
  • a vaccine composition is a composition designed to elicit an immune response to the recombinant bacterium, including any antigens that may be expressed by the bacterium.
  • the immune response is protective.
  • protecting means that the immune response contributes to the lessening of any symptoms associated with infection of a host with the pathogen the antigen was derived from or designed to elicit a response against.
  • a protective antigen from a pathogen such as Mycobacterium
  • the use of the term "protective" in this invention does not necessarily require that the host is completely protected from the effects of the pathogen.
  • Vaccine compositions of the present invention may be administered to any host capable of mounting an immune response.
  • hosts may include all vertebrates, for example, mammals, including domestic animals, agricultural animals, laboratory animals, and humans, and various species of birds, including domestic birds and birds of agricultural importance, such as chickens and turkeys.
  • the host is a warm-blooded animal.
  • the vaccine can be administered as a prophylactic or for treatment purposes.
  • the recombinant bacterium is alive when administered to a host in a vaccine composition of the invention.
  • Suitable vaccine composition formulations and methods of administration are detailed below.
  • a vaccine composition comprising a recombinant bacterium of the invention may optionally comprise one or more possible additives, such as carriers, preservatives, stabilizers, adjuvants, and other substances.
  • the vaccine comprises an adjuvant.
  • Adjuvants such as aluminum hydroxide or aluminum phosphate, are optionally added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response.
  • the use of a live attenuated recombinant bacterium may act as a natural adjuvant.
  • the vaccine compositions may further comprise additional components known in the art to improve the immune response to a vaccine, such as T cell co-stimulatory molecules or antibodies, such as anti-CTLA4. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences naturally found in bacteria, like CpG, are also potential vaccine adjuvants.
  • the vaccine may comprise a pharmaceutical carrier (or excipient).
  • a carrier may be any solvent or solid material for encapsulation that is non-toxic to the inoculated host and compatible with the recombinant bacterium.
  • a carrier may give form or consistency, or act as a diluent.
  • Suitable pharmaceutical carriers may include liquid carriers, such as normal saline and other non-toxic salts at or near physiological concentrations, and solid carriers not used for humans, such as talc or sucrose, or animal feed. Carriers may also include stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers.
  • the vaccine When used for administering via the bronchial tubes, the vaccine is preferably presented in the form of an aerosol.
  • Stabilizers such as lactose or monosodium glutamate (MSG) may be added to stabilize the vaccine formulation against a variety of conditions, such as temperature variations or a freeze-drying process.
  • the dosages of a vaccine composition of the invention can and will vary depending on the recombinant bacterium, the regulated antigen, and the intended host, as will be appreciated by one of skill in the art. Generally speaking, the dosage need only be sufficient to elicit a protective immune response in a majority of hosts. Routine experimentation may readily establish the required dosage. Typical initial dosages of vaccine for oral administration could be about 1 x10 7 to 1 x 10 10 CFU depending upon the age of the host to be immunized. Administering multiple dosages may also be used as needed to provide the desired level of protective immunity.
  • administration of the vaccine composition directly into the gut, nasopharynx, or bronchus is preferred, such as by oral administration, intranasal administration, gastric intubation or in the form of aerosols, although other methods of administering the recombinant bacterium, such as intravenous, intramuscular, subcutaneous injection or intramammary, intrapenial, intrarectal, vaginal administration, or other parenteral routes, are possible.
  • these compositions are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these compositions are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • recombinant bacterium may be administered orally. Oral administration of a composition comprising a recombinant bacterium allows for greater ease in disseminating vaccine compositions for infectious agents to a large number of people in need thereof, for example, in Third World countries or during times of biological warfare.
  • oral administration allows for attachment of the bacterium to, and invasion of, the gut-associated lymphoid tissues (GALT or Peyer's patches) and/or effective colonization of the mesenteric lymph nodes, liver, and spleen.
  • GALT gut-associated lymphoid tissues
  • This route of administration thus enhances the induction of mucosal immune responses as well as systemic and cellular immune responses.
  • recombinant bacterium may be administered by coarse spray.
  • the vaccines are administered by this whole-body spray route in an amount that is effective in eliciting an immune response, i.e. antibody and/or cellular immunity.
  • Whole-body spray administration is surprisingly effective for vaccines comprising a live avirulent derivative of an enteropathogenic bacteria such as attenuated Salmonella.
  • Spray administration of enteropathogenic bacteria avoids some of the disadvantages of other routes of administrations in that it does not require individual handling of chicks, it can be administered on day-of-hatch, and is easy to use under conditions normally found in commercial poultry production.
  • the effective doses, which elicit an immune response are roughly comparable to doses that are effective by the oral route of administration, such as administration in the drinking water.
  • kits comprising any one of the compositions above in a suitable aliquot for vaccinating a host in need thereof.
  • the kit further comprises instructions for use.
  • the composition is lyophilized such that addition of a hydrating agent (e.g., buffered saline) reconstitutes the composition to generate a vaccine composition ready to administer, preferably orally.
  • a hydrating agent e.g., buffered saline
  • a further aspect of the invention encompasses methods of using a recombinant bacterium of the invention.
  • the invention provides a method for modulating a host's immune system.
  • the method comprises administering to the host an effective amount of a composition comprising a recombinant bacterium of the invention.
  • an effective amount of a composition is an amount that will generate the desired immune response (e.g., mucosal, humoral or cellular).
  • Methods of monitoring a host's immune response are well-known to physicians, veterinarians, and other skilled practitioners. For instance, assays such as ELISA, and ELISPOT may be used.
  • Effectiveness may be determined by monitoring the amount of the antigen of interest remaining in the host, or by measuring a decrease in disease incidence caused by a given pathogen in a host.
  • cultures or swabs taken as biological samples from a host may be used to monitor the existence or amount of pathogen in the individual.
  • the invention provides a method for eliciting an immune response against a C. perfringens antigen in a host.
  • the method comprises administering to the host an effective amount of a composition comprising a recombinant bacterium of the invention.
  • a recombinant bacterium of the invention may be used in a method for eliciting an immune response against C. perfringens in a host in need thereof.
  • the method comprises administrating to the host an effective amount of a composition comprising a recombinant bacterium as described herein.
  • a recombinant bacterium described herein may be used in a method for ameliorating one or more symptoms of C. perfringens infection in a host in need thereof.
  • a recombinant bacterium may be used to ameliorate one or more symptoms of necrotic enteritis.
  • the method comprises administering an effective amount of a composition comprising a recombinant bacterium as described herein.
  • Example 1 Alpha-toxin antigen expressing recombinant bacteria
  • Clostridium perfringens CP995 and JGS4143 strains are type A strains originally isolated from the intestines of NE-affected chickens. CP995 was used for cloning the alpha toxin C-terminal domain, and JGS4143, (a hypervirulent strain), was used in the challenge experiments. Bacteria were grown in chopped meat broth (CMM) or brain heart infusion (BHI) medium with 0.04% D- cyclosehne (BBL, Franklin Lakes, NJ).
  • CCM chopped meat broth
  • BHI brain heart infusion
  • TSC Tryptose-sulfate- cycloserine
  • TSC-EY egg yolk
  • sheep blood agar were used for colony differentiation based on lecithinase or hemolytic activity, respectively.
  • Fluid thioglycolate medium (FTG) was used to cultivate large quantities of bacteria for animal inoculation and for bacterial resuscitation from tissue samples. All C. perfringens cultures were grown at 37°C under anaerobic condition using BBL GasPak systems. Both CP995 and JGS4143 were confirmed to be negative for ⁇ 2 toxin by PCR verification.
  • S. enterica serovar Typhimurium ⁇ 8914 containing defined attenuating deletions of the pabA and pabB genes, was used as the vaccine vector.
  • ⁇ 8914 is derived from a highly virulent S. enterica serovar Typhimurium strain ( ⁇ 3761 ).
  • ⁇ 8914 has an oral 50% lethal dose of greater than 1 x 10 9 CFU for 1 -day-old chicks, whereas the 50% lethal dose for the wild-type ⁇ 3761 is 3 x 10 3 CFU.
  • ⁇ 8914 also has a deletion in the gene encoding aspartate ⁇ -semialdehyde dehydrogenase ⁇ asd), which renders it deficient in the synthesis of diaminopimelic acid (DAP). Since DAP is an obligate component of the cell wall peptidoglycan, in the absence of exogenously supplied DAP the growth of ⁇ 8914 strictly depends on the complementation of the asd mutation with the vaccine antigen expression plasmid that carries the Salmonella asd gene. This dependence on Asd + plasmid complementation is the basis for the balanced lethal host-vector system, which abrogates the need for antibiotic resistance markers. Bacteria were grown at 37°C in Luha-Bertani (LB) culture medium containing 0.1 % dextrose. When required, 50 ⁇ g/ml DAP was added.
  • LB Luha-Bertani
  • C. perfhngens plcC gene Cloning of the C. perfhngens plcC gene.
  • C. perfringens DNA was isolated from a bacterial lysate prepared from colonies grown overnight on TSC agar plates. Bacteria were suspended in 150 ⁇ l 0.5 M NaOH, pH 8.5, incubated for 30 min at room temperature, and diluted with 25 ⁇ l Tris-CI, pH 7.4, and 425 ⁇ l water.
  • a 375-bp region of the alpha toxin gene (pic) encoding the carboxy-terminal end of the alpha toxin (amino acids 248 to 370) was PCR amplified from the CP995 lysate using standard PCR conditions with the following primer sequences: 5'- CGGAATTCGATCCATCAGTTGGAAAGAATGTA-3' (SEQ ID NO:1 ) and 5'- CCGAAGCTTATTATTTTATATTATAAGTTGAATTTCC-S' (SEQ ID NO:2).
  • rPlcC protein preparation The PCR product of plcC, the alpha toxin C-terminal domain-encoding sequence, was first cloned into the pBAD/HisB plasmid vector (Invitrogen, Carlsbad, CA) for the expression of a His-tagged recombinant PIcC (rPlcC) protein.
  • the plasmid pYA3910 (pBADHisB containing plcC) (Table 1 ) was electroporated into competent Escherichia coli Top10 cells (Invitrogen, Carlsbad, CA).
  • the expression of His-tagged rPlcC in the Top10 cells was induced by adding 0.02% L-arabinose into early-log-phase growing bacteria.
  • Bacteria were harvested from a 250-ml culture at an optical density at 600 nm (OD 6 oo) of 1.2 by centrifugation at 5,000 x g for 15 min.
  • the cell pellet was resuspended in 40 ml cell lysis solution (Sigma, St. Louis, MO), which contains lysozyme (0.2 mg/ml), benzonase (50 U/ml), and protease inhibitors. Following 15 min of incubation at room temperature in the lytic solution, the bacterial suspension was briefly sonicated (2 min) to ensure cell disruption.
  • Insoluble material was removed by centrifugation at 16,000 x g for 10 min.
  • the supernatant containing His-tagged protein was loaded onto 0.8- by 4-cm chromatography columns (Bio-Rad, Hercules, CA) packed with nickel-Sepharose gel (6%) (Sigma, St. Louis, MO).
  • the affinity gel matrix was washed with 50 mM NaH 2 PO 4 , pH 8.0, 0.3 M NaCI solution before and after the bacterial lysate was loaded. Proteins were eluted with 200 mM CsH 4 N 2 (imidazole) in the washing solution.
  • the elute was desalted and concentrated by a Centricon filtration system using 5,000 (5K) and 5OK nominal molecular weight membranes (Millipore, Billerica, MA). Protein was analyzed by electrophoresis on a 12% Tris-Bis gel (see Fig. 1 ) and by Western blotting using 6-HisG antibody (Invitrogen, Carlsbad, CA). The protein concentration was determined by a Bradford assay using bovine serum albumin as the standard. To produce rabbit anti- PIcC antibody, two rabbits were injected subcutaneously (s.c.) with 100 ⁇ g rPlcC protein emulsified in Freund's adjuvant. The rabbits were immunized three times, with 2-week intervals between injections, and the antiserum was tested for specific reactivity against rPlcC by immunoblot analysis (see Fig. 1 ).
  • the plasmids contain a modified P frc or P / pp promoter and a signal peptide sequence from E. coli class A ⁇ -lactamase (bla) or the outer membrane protein A (ompA) at the translation start site for the cloned antigen (Fig. 2).
  • the P trc or P /pp promoter directs the constitutive expression of the recombinant PIcC in Salmonella.
  • the signal peptides target the protein for secretion by the type Il secretion pathway into the periplasm, with subsequent release into the culture supernatant.
  • pYA3620 also has the ⁇ -lactamase C-terminal protein-coding sequence at the 3' end of the recombinant gene.
  • Such a fusion of the C-terminal peptide sequence of ⁇ -lactamase to the recombinant protein has been reported to facilitate the transport of recombinant protein across membranes.
  • PIcC expression plasmids pYA3977, pYA4110, and pYA4149 (Table 1 ) were propagated, and PIcC expression was confirmed first in E.
  • the plasmids were recovered from E. coli and introduced into competent ⁇ 8914 by electroporation.
  • the parent plasmid pYA3493 was similarly introduced into ⁇ 8914.
  • the vaccine strain harboring the expression plasmid was grown for more than 50 generations under nonselective conditions (i.e., in the presence of DAP) and then was tested for growth on medium without DAP.
  • RASV strains were grown overnight in 5 ml LB broth. The next day, cultures were inoculated into 100 ml LB broth and grown with aeration at 200 rpm until the culture reached an OD 6 oo ⁇ f 0.8.
  • rPlcC as a cytoplasmic soluble protein
  • 1 ml of the culture was transferred to a microcentrifuge tube and centhfuged for 3 min at 14,000 x g, and the pellet was resuspended in 500 ⁇ l Tris- HCI buffer (50 mM Tris-HCI, 100 mM NaCI, pH 7.2, 10 mM ⁇ -mercaptoethanol, and 2 mM phenylmethylsulfonyl fluoride).
  • Bacteria were lysed by sonication, and insoluble protein was removed by centrifugation at 14,00O x g for 2 min. The supernatant was tested for rPlcC.
  • bacteria were harvested from a 100-ml culture by centrifugation at 4,000 x g for 15 min at 4°C. Subcellularfractionation for periplasm contents was performed using the lysozyme digestion of the bacterial pellet by osmotic shock with sucrose as previously described. The culture supernatant was filtered through a 0.22- ⁇ m filter and concentrated by precipitation overnight at 4°C in a 10% trichloroacetic acid (TCA) solution. The protein samples were analyzed by SDS-PAGE and Western blotting.
  • TCA trichloroacetic acid
  • RASV inoculum preparation RASV strains from -80 0 C stock were spread on LB agar. Five colonies were inoculated into 5 ml broth and grown statically overnight at 37°C. The following day, the whole culture was inoculated into 100 ml prewarmed LB broth in a 500-ml culture flask and grown with constant shaking at 200 rpm to an OD 6 oo ⁇ f 0.8 (about 5 h of culture). Bacteria were harvested by centrifugation at 4,000 x g for 15 min at room temperature, and the pellet was resuspended in buffered saline with 1 % gelatin (BSG) solution.
  • BSG gelatin
  • the volume of the bacterial culture and BSG for resuspension was calculated to yield a bacterial concentration of 2 x 10 9 CFU/ml.
  • Chickens were inoculated orally with 0.5 ml of the suspension, which contained 1 x 10 9 CFU.
  • the chickens were administered 50 ⁇ g protein in a 100- ⁇ l volume suspension emulsified in incomplete Freund's adjuvant.
  • oral RASV priming followed by a parenteral protein boost immunization was carried out to evaluate the effect of primary immunization with RASV or a subunit vaccine on antibody titers.
  • RASV-3 was not included in this experiment.
  • Chickens were fed an antibiotic-free corn-based starter diet or a wheat/barley-based grower's diet (Purina Mills, St. Louis, MO). Feed and water were provided ad libitum. In the first week, chicks were reared at a brooding temperature of 32°C with 24 h of light. Subsequently, the cage temperature was kept at 25°C and the light schedule modified to 16 h of light and 8 h of dark.
  • perfringens was grown in 10 ml CMM broth for 18 h at 37°C, which then was inoculated into 1 liter of FTG and grown for 18 h.
  • the FTG culture was mixed with feed at a wt/vol ratio of 1 : 1.
  • the bacterial feed mix was freshly prepared twice per day and was provided to the chickens for four consecutive days.
  • the average number of bacteria in the 18-h FTG culture was 1 x 10 9 CFU/ml, and shortly after being mixed with feed, 10 7 CFU/g feed was recovered, which declined to 10 2 to 10 3 CFU/g feed after 10 h on the feeder.
  • One day after the end of the challenge infection five birds were euthanized by CO2 inhalation for postmortem examination. The remaining five chickens were used for delayed-type hypersensitivity (DTH) assays and euthanized five days later. Individual body weights were measured before and after challenge infections.
  • DTH delayed-type hypersensitivity
  • the cloacal swab preparations also were spread on TSC-EY agar plates and incubated in an anaerobic jar to identify lecithinase-positive (C. perfringens) colonies. After challenge infection with C. perfringens, segments of the ileum and cecum with the intestinal contents were aseptically collected and homogenized in BSG. Tissue homogenates were diluted in BSG and spread on MacConkey agar plates for S. enterica serovar Typhimurium detection and on TSC agar plates for C. perfringens detection.
  • each well's contents was measured at 405 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). A volume of 100 ⁇ l/well of the test samples, antibodies, or washing solutions was used in each steps. The antibody response in the serum also was tested by the immunoblot analysis of immunized chicken sera against alpha toxin obtained from concentrated culture supernatant of C. perfringens and rPlcC proteins.
  • Alpha toxin neutralization test The neutralization of alpha toxin by serum antibody was determined by the inhibition of red blood cell (RBC) hemolysis by alpha toxin. RBCs were prepared by washing samples of freshly collected rabbit blood twice in PBS and diluting them to 2% (vol/vol) in PBS containing 3 mM CaCI 2 . Alpha toxin was obtained from the supernatant of a C. perfringens culture grown overnight by sequential filtration through 100- and 10-kDa Amicon Ultra filters (Millipore, Billerica, MA).
  • RBC red blood cell
  • the enzymatic activity of the culture supernatant concentrate was evaluated and quantified using a phosphatidylcholine-phospholipase C assay kit (Invitrogen, Carlsbad, CA).
  • the concentrated culture supernatant was diluted in PBS containing 0.1 mM CaCI 2 , and 100- ⁇ l aliquots containing an estimated 250 ng protein (about 200 U) were distributed into a 96-well dilution plate.
  • Purified recombinant alpha toxin (250 ng) (Sigma, St. Louis, MO) was included as a control.
  • Test sera (pooled serum samples) were twofold diluted in PBS (1 :10, 1 :20, and 1 :40), and 100 ⁇ l was added into wells.
  • Control wells contained either serum with no toxin or alpha toxin alone. Well contents were mixed and incubated for 1 h at 37°C with slow agitation. After 1 h, 100 ⁇ l of the 2% RBC solution was added into each well. After incubation for 2 h at 37°C, plates were chilled for 15 min at 4°C and briefly centhfuged at 500 x g to sediment intact cells (i.e., RBCs). The absorbance of well contents was measured at 540 nm using a microtiter plate reader.
  • DTH assay Four weeks after the RASV boost immunization and 3 days after the challenge infection, five chickens per group were injected intradermal ⁇ with 20 ⁇ g of the rPlcC protein in 50 ⁇ l saline into the left footpad at the toe web between the first and second digits. The right foot was injected with sterile saline as a negative control. The thickness of the toe web was measured with a digital micrometric caliper at 48 and 72 h after antigen or saline injection. Data were expressed as the difference between results for the left foot and the right foot (control).
  • Serum bacteriostatic (growth inhibition) assay Sera from immunized or control chickens were filtered through 0.45- ⁇ m pore filters, decomplemented by being heated at 56°C in a water bath for 30 min, and added into the CMM broth culture at 1 :20 and 1 :40 final dilutions. A volume of 100 ⁇ l overnight culture of C. perfringens diluted to a concentration of 10 5 CFU per ml was added into 1 ml of culture medium containing serum, and the cultures were incubated anaerobically at 37°C. The growth of bacteria was monitored by the culture OD, and the number of CFU was monitored by plating serial dilutions on TSC-EY agar plates at 12 and 18 h.
  • the suspension (10 ml) was centhfuged at 5,000 xg for 15 min, and the pellet was washed twice and resuspended in PBS. Live cells were spread on slides from colonies and were killed and fixed by being heated. Slides were dried and covered with PBS containing 1 % bovine serum albumin and incubated for 1 h at room temperature in a humidified box. The blocking solution was removed, and 200 ⁇ l of the test serum from immunized or control chickens diluted 1 :50 in PBS was spread onto the slide and covered with a coverslip. Slides were incubated overnight at 4°C.
  • alpha toxin The primary structure of alpha toxin from NE isolate CP995 shows 98% sequence identity to the type A reference strain ATCC 13124 (GenBank accession no. M24904), with a few amino acid substitutions (Leui 6 to Thr, Lysi 7 to GIn, Alai 74 to Asp, ThH 77 to Pro, Ser 3 35 to Pro, Gly 3 63 to Arg, and Asn 364 to Lys) and a deletion of lle 2 i (Fig. 3). Except for the absence of lle 2 i, the amino acid substitutions at the specific sites are common variations among type A isolates from humans and animals. These variations do not affect alpha toxin's phospholipase C and hemolytic activities or hinder the neutralizing ability of C terminus- specific antibody against alpha toxin.
  • PIcC expression in S. enterica serovar Typhimurium vaccine vector The expression of the PIcC protein in the S. enterica serovar Typhimurium vaccine strain was detected on the SDS-PAGE gel stained with Coomassie blue (Fig. 1 ). High levels of PIcC expression were obtained with all three plasmid constructs. The PIcC protein concentration was estimated at roughly 100 ng/ ⁇ l bacterial lysate, as measured by a densitometry analysis that compared the results to those for a standard concentration of bovine serum albumin (ChemiDoc XRS; Bio-Rad, Hercules, CA). PIcC was effectively secreted by the S.
  • enterica serovar Typhimurium vaccine strains through a type Il secretion system.
  • the fusion with the OmpA or BIa signal peptide sufficiently enabled the transport of PIcC into the periplasm, which then was secreted across the outer membrane into the culture supernatant.
  • No ⁇ -galactosidase was detected in the culture supernatant of the RASV ⁇ 8914 (pYA9977) afrS73::Mud J strain, thus indicating that bacterial lysis had not occurred.
  • the molecular masses of PIcC expressed from the three constructs appear different due to differences in the sizes of the signal peptides, the linker amino acids, and the ⁇ -lactamase C-terminal fusion in pYA4149 constructs (Fig. 2). Accordingly, the molecular mass of PIcC was 16 kDa in ⁇ 8914(pYA3977), 14 kDa in ⁇ 8914(pYA4110), and 18 kDa in ⁇ 8914(pYA4149). The molecular mass of the His- tagged rPlcC was 19 kDa.
  • RASV-immunized chickens generally had low serum antibody responses, as measured by ELISA, at serum dilutions as low as 1 :40. Unexpectedly, chickens that received s.c. immunization with rPlcC also showed a low serum IgG response similar to that of RASV-immunized chickens. Formerly, we tested whether RASV or rPlcC immunization gives some priming effect when followed by a boost protein immunization at a later time point.
  • Sera from the RASV-immunized chickens were tested by Western blotting on SDS-PAGE-separated rPlcC and the whole alpha toxin, and both PIcC protein and the complete alpha toxin obtained from the culture supernatant of C. perfringens were detected (Fig. 5). This confirmed the presence of serum antibody responses in RASV-immunized chickens. Sera from the RASV- or rPlcC-vaccinated chickens reacted positively with the whole alpha toxin, whether it was denatured by TCA precipitation or not (i.e., concentrated by filtration) prior to separation on SDS-PAGE.
  • enterica serovar Typhimurium replicates intracellular ⁇ within antigen-presenting cells, it presumably facilitates the delivery of recombinant antigens by the endogenous antigen presentation pathway to prime CD4 + T cells and the cell-mediated response.
  • DTH is commonly used as an index for cellular responses.
  • Alpha toxin neutralization Toxin neutralization by serum antibody was measured as a function of alpha toxin-induced RBC lysis. Pooled sera from chickens immunized with RASV or rPlcC inhibited the hemolytic effect of the alpha toxin in a C. perfringens culture supernatant (Fig.7). The highest level of RBC lysis inhibition was observed with sera from birds vaccinated with rPlcC and RASV-3 [ ⁇ 8914(pYA4149)], with inhibitions of 76 and 71 %, respectively.
  • the S. enterica serovar Typhimurium vaccine strain was readily detected in cloacal swabs collected 1 week after the boost immunization and in the intestinal segments (from the ileum and cecum) collected during autopsy at 3 weeks after immunization.
  • the average S. enterica serovar Typhimurium numbers in cloacal swabs were 1 x 10 4 CFU/g of fecal material, and 3 weeks later the numbers in the ileum and in the cecum were, on average, 1 x 10 2 and 1 x 10 4 CFU per gram of tissue, respectively.
  • No S. enterica serovar Typhimurium was detected in the liver or spleen at 3 weeks after immunization.
  • C. perfringens could not be detected from cloacal swab samples collected prior to the challenge infection.
  • C. perfringens resident flora cells are identified by culture from healthy young birds, especially in birds reared under experimental conditions. After the challenge infection, generally smaller numbers of C. perfringens were detected from intestines, even at 1 day after the challenge infection. The average numbers of C. perfringens cells detected in the intestines were less than 1 x 10 3 and 1 x 10 5 CFU/g in the ileum and cecum, respectively.
  • hematoxylin- and eosin- stained sections also revealed clusters of C. perfringens in the intestinal lumen and mucosal surfaces that were seldom attached to epithelial surfaces (Fig. 9E).
  • Sites with bacterial clumps often depicted a mild defect of the epithelial surface and microvilli mostly in nonimmunized or control RAS-immunized chickens.
  • the lesions were severe and more frequent in nonimmunized (control) chickens, while immunized chickens had minimal lesions in a few chickens (Table 3).
  • the histopathological lesions had receded by 4 days after challenge.
  • RASV-immunized chickens showed significantly higher percentages of body weight gain at 10 days after challenge with C. perfringens compared to the average body weight gain for nonimmunized chickens (Table 3).
  • a subunit vaccine consisting of the C-terminal domain of alpha toxin
  • PIcC protects mice against gas gangrene.
  • no injectable PIcC subunit vaccine or live attenuated S. ente ⁇ ca serovar Typhimurium-delivered PIcC vaccine to induce protective immunity against C. perfringens type A strain-caused enteric disease has been evaluated before.
  • the current study showed that an RASV expressing PIcC or an rPlcC subunit vaccine induced toxin-neutralizing antibodies and reduced intestinal lesion development and body weight loss in chickens challenged with virulent C. perfringens.
  • many investigators showed protection against NE by vaccination with a toxoid vaccine that consisted of an inactivated whole alpha toxin.
  • the netB gene encodes a protein that shows limited homology with
  • C. perfringens ⁇ -toxin It is found primarily in C. perfringens isolated from birds suffering from necrotic enteritis, but not from healthy animals. NetB is cytotoxic to chicken leghorn male hepatoma cells in vitro and it is required for the induction of necrotic enteritis symptoms in chickens. We have cloned the naturally occurring netB gene into a number of our Asd+ expression vectors and are performing immunological evaluations on Salmonella vaccine strains carrying these plasmids.
  • Example 3 Additional protective antigens for expression in recombinant attenuated Salmonella vaccines.
  • Additional C. perfringens antigens that provide protection against necrotic enteritis when administered to chickens by injection into muscle tissue or when delivered by attenuated Salmonella include glyceraldehyde 3-phosphate dehydrogenase (GDP), pyruvate:ferredoxin oxidoreductase (PFOR), truncated pyruvate:ferredoxin oxidoreductase (tPFOR), fructose 1 ,6-biphosphate aldolase (FBA), a hypothetical protein (HP), and a truncated form of hypothetical protein (tHP).
  • GDP glyceraldehyde 3-phosphate dehydrogenase
  • PFOR pyruvate:ferredoxin oxidoreductase
  • tPFOR truncated pyruvate:ferredoxin oxidoreductase
  • FBA fructose 1 ,6-biphosphate aldolase
  • the optimized genes are fused to a secretion signal such as bla SS, ompA SS, bla SS + CT, eltll-B SS or dsbA SS by cloning the optimized gene into AsdA+ expression plasmid pYA3493, pYA4101 , pYA3620, pYA4531 or pYA4538, respectively and expressed in an attenuated S. Typhimurium strain such as ⁇ 9352.
  • Example 4 Expression of two or more antigens in a single Salmonella vaccine strain.
  • a bla SS-p/cC fusion gene and a bla SS-netB fusion gene are cloned into the same Asd+ plasmid vector as an operon fusion.
  • Expression is driven from a single, regulatable promoter, such as the P trc promoter. Both genes are expressed in the same attenuated Aasd Salmonella strain in amounts adequate to induce a protective immune response.
  • each gene is transcribed from its own, regulatable promoter.
  • each gene is cloned into a different expression plasmid, one with an Asd+ selectable marker and the other with a different selectable marker, such as DadB+.
  • the bla SS netB (optimized) fusion protein is cloned into DadB+ expression vector pYA4555 ( Figure 20) to yield pYA4555 + bla SS netB (optimized) ( Figure 21 ).
  • the bla SS netB (optimized) gene is transcribed from the regulatable P22 P L promoter.
  • Plasmid pYA4555 + bla SS netB (optimized) and plasmid pYA4679 ( Figure 22) are introduced into strain such as ⁇ 9590 ⁇ Apmi-2426 A ⁇ gmd-fcl)-26 ⁇ P fur8 i::TT araC P BAD fur ⁇ P cr p527::TT araC P BAD crp AasdA27::TT araC P BAD C2 AaraE25 AaraBAD23 ArelA198::araC P BAD lad TT AsopB1925 AagfBAC811 Aalr-3 AdadB4), which carries deletions in the asdA, alr-3 and dadB4 genes, allowing for selection of both AsdA+ plasmids and DadB+ plasmids.
  • this strain produces both the C2 repressor and the Lacl repressor, each transcribed from its own arabinose-regulated P BAD promoter, providing regulatable expression of netB from the P22 P L promoter (pYA4555 + bla SS netB (optimized)) and plcC from the P tr c promoter (pYA4679), respectively.
  • Two antigens, PIcC and NetB are synthesized in a single bacterium in amounts adequate to induce an immune response against C. perf ⁇ ngens.
  • Other attenuated Salmonella strains that can support the presence of two plasmids may also be used.
  • Other plasmid selectable markers that are used include murA, aroA, aroC, aroD, HvC or HvE. Plasmids carrying these markers are maintained in strains carrying a deletion of the selectable marker gene.
  • the genes for other protective antigens are codon-optimized and optimized for G+C content are used.
  • the genes encoding tPFOR (Fig. 16), tHP (Fig. 17), GDP (Fig. 18) or FBA (Fig. 19) are cloned into separate plasmids or as operon fusions or as protein fusions and are expressed from one or more plasmids.
  • one plasmid carries an operon fusion of two antigens, such as plcC and netB, and another plasmid directs the synthesis of a third antigen, such as tPFOR.
  • Example 5 Expression of one or more antigens host-adapted Salmonella.
  • Clostridium perfringens causes necrotizing enteritis in the small intestines of a variety of species, including cattle (Jejunal hemorrhagic syndrome, necrotic enteritis), swine and even humans, which occurs sporadically in underdeveloped countries. Some factors that predispose to C. perfringens induced necrotic enteritis include protozoan and helminth infections.
  • C. perfringens type A associated diarrhea is one of the top 5 causes of food borne bacterial diarrheal disease ranked by CDC in the U.S. ⁇ -toxin is particularly responsible for sublethal effects on enterocytes that could lead to malabsorption and stunting in children in developing countries.
  • Host-adapted Salmonella may be attenuated and further modified for this purpose as described above.
  • Attenuated Salmonella Typhi and Salmonella Paratyphi A can be used as the antigen delivery vector for a human vaccine
  • attenuated Salmonella Choleraesuis can be used for a swine vaccine
  • attenuated Salmonella Dublin can be used for a bovine vaccine.
  • attenuated Salmonella Gallinarum may be used for a poultry vaccine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention a trait à des bactéries recombinantes et à des compositions immunogènes comprenant lesdites bactéries. Ladite composition immunogène peut être utilisée pour induire une réponse immunitaire contre C. perfringens.
PCT/US2008/078993 2007-10-05 2008-10-06 Bactérie recombinante capable de susciter une réponse immunitaire protectrice contre c. perfringens WO2009046451A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/681,711 US9040059B2 (en) 2007-10-05 2008-10-06 Recombinant bacterium capable of eliciting a protective immune response against C. perfringens

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US97808407P 2007-10-05 2007-10-05
US60/978,084 2007-10-05

Publications (1)

Publication Number Publication Date
WO2009046451A1 true WO2009046451A1 (fr) 2009-04-09

Family

ID=40526735

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2008/078993 WO2009046451A1 (fr) 2007-10-05 2008-10-06 Bactérie recombinante capable de susciter une réponse immunitaire protectrice contre c. perfringens
PCT/US2008/078991 WO2009046449A1 (fr) 2007-10-05 2008-10-06 Bactéries recombinantes capables de susciter une réponse immunitaire contre des agents pathogènes entériques

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US2008/078991 WO2009046449A1 (fr) 2007-10-05 2008-10-06 Bactéries recombinantes capables de susciter une réponse immunitaire contre des agents pathogènes entériques

Country Status (2)

Country Link
US (2) US9040059B2 (fr)
WO (2) WO2009046451A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8445254B2 (en) 2007-05-10 2013-05-21 The Arizona Board Of Regents For And On Behalf Of Arizona State University Regulated expression of antigen and/or regulated attentuation to enhance vaccine immunogenicity and/or safety
US8465755B2 (en) 2007-10-05 2013-06-18 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium capable of eliciting an immune response against enteric pathogens
US8889121B2 (en) 2010-01-22 2014-11-18 The Arizona Board Of Regents For An On Behalf Of Arizona State University Bacterium comprising a regulated rfaH nucleic acid
US9045742B2 (en) 2009-05-29 2015-06-02 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant Edwardsiella bacterium
US9050285B2 (en) 2008-10-17 2015-06-09 The United States of America National Institutes of Health (NH), U.S. Dept. of Health and Human Services (DHHS) Recombinant bacterium capable of eliciting an immune response against Streptococcus pneumoniae
US9062297B2 (en) 2010-01-13 2015-06-23 The Arizona Board Of Regents For And On Behalf Of Arizona State University Yersinia pestis vaccine
US9163219B2 (en) 2009-04-14 2015-10-20 Arizona Board Of Regents On Behalf Of Arizona State University Single expression vector for generation of a virus with a segmented genome
US9303264B2 (en) 2012-05-18 2016-04-05 The Arizona Board Of Regents For And On Behalf Of Arizona State University Photosynthetic microorganisms expressing thermostable lipase
US9481888B2 (en) 2009-05-22 2016-11-01 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium and methods of antigen and nucleic acid delivery
US9580718B2 (en) 2013-06-17 2017-02-28 Arizona Board Of Regents On Behalf Of Arizona State University Attenuated live bacteria with increased acid resistance and methods of use thereof
US9598697B2 (en) 2010-05-28 2017-03-21 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium to decrease tumor growth
EP3661547A4 (fr) * 2017-08-04 2021-06-09 University of Florida Research Foundation, Incorporated Induction d'une immunité protectrice contre des antigènes
US11154606B2 (en) 2017-07-05 2021-10-26 Arizona Board Of Regents On Behalf Of Arizona State University Vaccine for prevention of necrotic enteritis in poultry
US11788056B2 (en) 2017-01-23 2023-10-17 University Of Florida Research Foundation, Incorporated Induction of protective immunity against antigens

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080124355A1 (en) 2006-09-22 2008-05-29 David Gordon Bermudes Live bacterial vaccines for viral infection prophylaxis or treatment
US20100317084A1 (en) * 2007-05-10 2010-12-16 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacteria comprising vectors for expression of nucleic acid sequences encoding antigens
RU2474587C2 (ru) * 2007-06-08 2013-02-10 Острэйлиан Паултри Срс Пти Лтд КЛОСТРИДИАЛЬНЫЙ ТОКСИН NetB
US10174086B2 (en) 2011-10-25 2019-01-08 University Of Exeter Vaccine
GB201118394D0 (en) 2011-10-25 2011-12-07 Univ Exeter Vaccine
ES2708430T3 (es) * 2012-03-22 2019-04-09 Km Biologics Co Ltd Vacuna de LPS
NZ700199A (en) 2012-03-29 2016-08-26 Univ Exeter Epsilon toxin epitopes from clostridium perfringens with reduced toxicity
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria
US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
CN110520153B (zh) * 2017-01-27 2024-04-02 佛罗里达大学研究基金公司 在禽类中控制肠道沙门氏菌和减少弯曲杆菌的食品安全疫苗
US11254713B2 (en) 2017-05-01 2022-02-22 Arizona Board Of Regents On Behalf Of Arizona State University Method to enhance immunogenicity of live typhoid vaccines and carriers
US11541105B2 (en) 2018-06-01 2023-01-03 The Research Foundation For The State University Of New York Compositions and methods for disrupting biofilm formation and maintenance
US20230123485A1 (en) * 2020-03-11 2023-04-20 Superbrewed Food, Inc. Microbial compositions and uses thereof
US10973908B1 (en) 2020-05-14 2021-04-13 David Gordon Bermudes Expression of SARS-CoV-2 spike protein receptor binding domain in attenuated salmonella as a vaccine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040101531A1 (en) * 2002-04-16 2004-05-27 Roy Curtiss Immunogenic compositions and vaccines comprising carrier bacteria that secrete antigens
US20060171917A1 (en) * 2004-12-02 2006-08-03 Campbell Robert L Vaccine formulations for intradermal delivery comprising adjuvants and antigenic agents

Family Cites Families (68)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4190495A (en) 1976-09-27 1980-02-26 Research Corporation Modified microorganisms and method of preparing and using same
US4968619A (en) * 1976-09-27 1990-11-06 Research Corporation Modified microorganisms and method of preparing and using same
US5210035A (en) * 1980-05-19 1993-05-11 Board Of Trustees Of Leland Stanford Jr. University Non-reventing live vaccines
US4888170A (en) * 1981-10-22 1989-12-19 Research Corporation Vaccines obtained from antigenic gene products of recombinant genes
US5855880A (en) 1987-06-04 1999-01-05 Washington University Avirulent microbes and uses therefor
US5387744A (en) * 1987-06-04 1995-02-07 Washington University Avirulent microbes and uses therefor: Salmonella typhi
US5294441A (en) 1987-06-04 1994-03-15 Washington University Avirulent microbes and uses therefor: salmonella typhi
US5468485A (en) 1987-06-04 1995-11-21 Washington University Avirulent microbes and uses therefor
IL86583A0 (en) 1987-06-04 1988-11-15 Molecular Eng Ass Vaccine containing a derivative of a microbe and method for the production thereof
ATE121785T1 (de) 1987-10-07 1995-05-15 Univ Washington Verfahren zur erhaltung eines erwünschten rekombinanten gens in einer genetischen zellpopulation.
WO1990002484A1 (fr) * 1988-09-06 1990-03-22 Washington University Immunisation par voie orale a l'aide de plantes transgeniques
WO1990011687A1 (fr) 1989-03-31 1990-10-18 Washington University VACCINS CONTENANT DES MICROORGANISMES AVIRULENTS DU TYPE phoP
WO1990011688A1 (fr) 1989-03-31 1990-10-18 Washington University Microbes avirulents et leurs emplois
JPH04504656A (ja) 1989-03-31 1992-08-20 ワシントン ユニバーシティー ボルデテラワクチン
CA2072633A1 (fr) 1989-11-03 1991-05-04 Roy Curtiss Iii Vaccins a protection croisee a base de salmonelles
ATE234917T1 (de) 1990-11-09 2003-04-15 Univ Washington Avirulente salmonella mikroben mit einer mutation in dem cdt gen und ihre verwendung
AU9094191A (en) 1990-11-21 1992-06-25 Washington University Recombinant avirulent salmonella antifertility vaccines
TW201794B (fr) 1991-05-03 1993-03-11 American Cyanamid Co
WO1993004202A1 (fr) 1991-08-22 1993-03-04 Washington University Sondes de polynucleotide de detection de salmonelle
US5817317A (en) * 1992-05-20 1998-10-06 The Secretary Of State For Defense Of Great Britian & Northern Ireland Clostridium perfringens vaccines
US5536658A (en) 1992-10-23 1996-07-16 The University Of Georgia Research Foundation Chondroitinase attenuated Edwardsiella ictaluri and a vaccine for prevention of enteric septicemia (es) in fish
WO1994024291A2 (fr) 1993-04-15 1994-10-27 Washington University Compositions de salmonelle recombinee contenant des antigenes, leur utilisation dans des vaccins antipaludiques et leur procede de preparation
ITPD940054A1 (it) * 1994-03-23 1995-09-23 Fidia Advanced Biopolymers Srl Polisaccaridi solfatati
DE69635579D1 (de) * 1995-06-07 2006-01-19 Univ St Louis Rekombinant bakterielle system mit umweltbeschränkte lebensfähigkeit
US6190657B1 (en) * 1995-06-07 2001-02-20 Yale University Vectors for the diagnosis and treatment of solid tumors including melanoma
US5780448A (en) * 1995-11-07 1998-07-14 Ottawa Civic Hospital Loeb Research DNA-based vaccination of fish
GB9605222D0 (en) * 1996-03-12 1996-05-15 Secr Defence Clostridium perfringens epsilon toxin vaccines
US5827705A (en) 1996-03-22 1998-10-27 South Alabama Medical Science Foundation Molecule and method for importing DNA into a nucleus
AU7105598A (en) * 1997-04-11 1998-11-11 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College, The Attenuated, invasive vaccines against fish pathogens
US6024961A (en) 1997-11-14 2000-02-15 Washington University Recombinant avirulent immunogenic S typhi having rpos positive phenotype
US6248329B1 (en) * 1998-06-01 2001-06-19 Ramaswamy Chandrashekar Parasitic helminth cuticlin nucleic acid molecules and uses thereof
US6399074B1 (en) * 1998-07-24 2002-06-04 Megan Health, Inc. Live attenuated salmonella vaccines to control avian pathogens
CA2341349C (fr) 1998-09-04 2013-12-10 Creatogen Aktiengesellschaft Cellules attenuees renfermant des mutants du sp12, porteurs et composititions contenant ces derniers, methodes de production et utilisations
CZ20011538A3 (cs) 1998-12-02 2001-11-14 University Of Maryland, Baltimore Nezávisle fungující expresní kazeta, amplifikovatelný plazmidový replikon, bakteriální buňka, vakcinační vektor, podmíněně nestabilní plazmid, způsob vyvolání imunitní odpovědi, DNA a expresní plazmid
US6780405B1 (en) 2000-04-28 2004-08-24 Avant Immunotherapeutics, Inc. Regulated antigen delivery system (RADS)
US6872547B1 (en) 2000-10-11 2005-03-29 Washington University Functional balanced-lethal host-vector systems
CA2463482A1 (fr) 2000-10-12 2003-04-18 Washington University Of St. Louis Microbes a mutation d'attenuation renfermant un terminateur de transcription
US20040137003A1 (en) 2001-04-30 2004-07-15 Curtiss Iii Roy Regulated antigen delivery system (rads)
ES2192949B1 (es) 2001-07-31 2004-08-16 Universidad De Zaragoza. Disminucion de la virulencia de mycobacterium tuberculosis por la inactivacion del gen phop.
US7294504B1 (en) 2001-12-27 2007-11-13 Allele Biotechnology & Pharmaceuticals, Inc. Methods and compositions for DNA mediated gene silencing
GB0206359D0 (en) 2002-03-18 2002-05-01 Glaxosmithkline Biolog Sa Viral antigens
US7655241B2 (en) 2002-03-21 2010-02-02 The Board Of Regents Of The University Of Texas System Methods and compositions for vaccination against or involving enterobacteriaceae bacteria
US7195757B2 (en) 2002-04-15 2007-03-27 Washington University Modulation of immune responses to foreign antigens expressed by recombinant attenuated bacterial vectors
CA2526895A1 (fr) 2002-04-15 2003-11-27 Washington University Attenuation regulee de vaccins vivants permettant d'ameliorer l'immunogenicite protectrice croisee
AU2003228603A1 (en) 2002-04-22 2003-11-10 Rtc Research And Development, Llc. Compositions and methods for promoting weight loss, thermogenesis, appetite suppression, lean muscle mass, increasing metabolism and boosting energy levels, and use as a dietary supplement in mammals
ATE318916T1 (de) 2002-09-01 2006-03-15 Univ Washington Kontrollierte bakterielle lyse zur verabreichung von dna vakzinvektoren und impfantigen
US9453251B2 (en) * 2002-10-08 2016-09-27 Pfenex Inc. Expression of mammalian proteins in Pseudomonas fluorescens
AR042015A1 (es) 2002-11-08 2005-06-08 Avant Immunotherapeutics Inc Marcador genetico relacionado con la capacidad de producir acido sulfhidrico para vacunas bacterianas atenuadas
US7695725B2 (en) 2003-02-06 2010-04-13 Aduro Biotech Modified free-living microbes, vaccine compositions and methods of use thereof
EP1618193B1 (fr) * 2003-04-16 2008-05-07 BASF Plant Science GmbH Utilisation de genes pour augmenter la teneur en huile dans les plantes
ATE420160T1 (de) * 2003-06-18 2009-01-15 Genelux Corp Modifizierte rekombinante vacciniaviren, verwendungen davon
US7390646B2 (en) 2003-09-17 2008-06-24 The Regents Of The University Of California Bacterial vectors and methods of use thereof
US7638485B2 (en) * 2003-12-02 2009-12-29 Cleveland Biolabs, Inc. Modulating apoptosis
US20080096809A1 (en) 2003-12-22 2008-04-24 Yeda Research & Development Co. Ltd. Diastereomeric Peptides Useful as Inhibitors of Membrane Protein Assembly
KR20070086344A (ko) * 2004-11-19 2007-08-27 위스콘신 얼럼나이 리서어치 화운데이션 탠덤 전사 유닛을 갖는 재조합 인플루엔자 벡터
US20100317084A1 (en) 2007-05-10 2010-12-16 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacteria comprising vectors for expression of nucleic acid sequences encoding antigens
WO2009025888A2 (fr) 2007-05-10 2009-02-26 The Arizona Board Of Regents For And On Behalf Of Arizona State University Expression régulée d'antigène et/ou atténuation régulée pour accroître l'immunogénicité et/ou la sécurité de vaccins
CA2685533C (fr) * 2007-06-22 2019-07-02 University Of Guelph Vaccin contre <i>clostridium perfringens</i>
WO2009046451A1 (fr) 2007-10-05 2009-04-09 The Arizona Board Of Regents For And On Behalf Of Arizona State University Bactérie recombinante capable de susciter une réponse immunitaire protectrice contre c. perfringens
US7998461B2 (en) * 2007-11-15 2011-08-16 University Of Massachusetts Salmonella cancer therapeutics and related therapeutic methods
WO2010045620A1 (fr) 2008-10-17 2010-04-22 The Arizona Board Of Regents For And On Behalf Of Arizona State University Bactérie recombinante pouvant éliciter une réponse immunitaire contre le streptococcus pneumoniae
CN110129296A (zh) 2008-12-23 2019-08-16 Reg生命科学有限责任公司 硫酯酶相关的方法和组合物
WO2010078584A1 (fr) 2009-01-05 2010-07-08 Arizona Board Of Regents Cyanobactérie produisant des lipides neutres
US9163219B2 (en) * 2009-04-14 2015-10-20 Arizona Board Of Regents On Behalf Of Arizona State University Single expression vector for generation of a virus with a segmented genome
WO2010135563A1 (fr) 2009-05-22 2010-11-25 The Arizona Board Of Regents For And On Behalf Of Arizona State University Bactérie de recombinaison et méthodes d'administration d'antigène et d'acide nucléique
US8889121B2 (en) 2010-01-22 2014-11-18 The Arizona Board Of Regents For An On Behalf Of Arizona State University Bacterium comprising a regulated rfaH nucleic acid
US9598697B2 (en) 2010-05-28 2017-03-21 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium to decrease tumor growth
US9198950B2 (en) 2010-11-24 2015-12-01 Arizona Board Of Regents On Behalf Of Arizona State University Recombinant bacterium comprising a toxin/antitoxin system

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040101531A1 (en) * 2002-04-16 2004-05-27 Roy Curtiss Immunogenic compositions and vaccines comprising carrier bacteria that secrete antigens
US20060171917A1 (en) * 2004-12-02 2006-08-03 Campbell Robert L Vaccine formulations for intradermal delivery comprising adjuvants and antigenic agents

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8445254B2 (en) 2007-05-10 2013-05-21 The Arizona Board Of Regents For And On Behalf Of Arizona State University Regulated expression of antigen and/or regulated attentuation to enhance vaccine immunogenicity and/or safety
US11920139B2 (en) 2007-05-10 2024-03-05 The Washington University Regulated expression of antigen and/or regulated attenuation to enhance vaccine immunogenicity and/or safety
US11180765B2 (en) 2007-05-10 2021-11-23 The Arizona Board Of Regents For And On Behalf Of Arizona State University Regulated expression of antigen and/or regulated attenuation to enhance vaccine immunogenicity and/or safety
US10774334B2 (en) 2007-05-10 2020-09-15 Arizona Broad of Regents on Behalf of Arizona State University Regulated expression of antigen and/or regulated attenuation to enhance vaccine immunogenicity and/or safety
US9297015B2 (en) 2007-05-10 2016-03-29 Arizona Board Of Regents For And On Behalf Of Arizona State University Regulated expression of antigen and/or regulated attentuation to enhance vaccine immunogenicity and/or safety
US8465755B2 (en) 2007-10-05 2013-06-18 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium capable of eliciting an immune response against enteric pathogens
US9040059B2 (en) 2007-10-05 2015-05-26 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium capable of eliciting a protective immune response against C. perfringens
US9050285B2 (en) 2008-10-17 2015-06-09 The United States of America National Institutes of Health (NH), U.S. Dept. of Health and Human Services (DHHS) Recombinant bacterium capable of eliciting an immune response against Streptococcus pneumoniae
US9163219B2 (en) 2009-04-14 2015-10-20 Arizona Board Of Regents On Behalf Of Arizona State University Single expression vector for generation of a virus with a segmented genome
US9481888B2 (en) 2009-05-22 2016-11-01 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium and methods of antigen and nucleic acid delivery
US9045742B2 (en) 2009-05-29 2015-06-02 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant Edwardsiella bacterium
US9062297B2 (en) 2010-01-13 2015-06-23 The Arizona Board Of Regents For And On Behalf Of Arizona State University Yersinia pestis vaccine
US8889121B2 (en) 2010-01-22 2014-11-18 The Arizona Board Of Regents For An On Behalf Of Arizona State University Bacterium comprising a regulated rfaH nucleic acid
US9598697B2 (en) 2010-05-28 2017-03-21 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium to decrease tumor growth
US9303264B2 (en) 2012-05-18 2016-04-05 The Arizona Board Of Regents For And On Behalf Of Arizona State University Photosynthetic microorganisms expressing thermostable lipase
US9580718B2 (en) 2013-06-17 2017-02-28 Arizona Board Of Regents On Behalf Of Arizona State University Attenuated live bacteria with increased acid resistance and methods of use thereof
US11788056B2 (en) 2017-01-23 2023-10-17 University Of Florida Research Foundation, Incorporated Induction of protective immunity against antigens
US11154606B2 (en) 2017-07-05 2021-10-26 Arizona Board Of Regents On Behalf Of Arizona State University Vaccine for prevention of necrotic enteritis in poultry
EP3661547A4 (fr) * 2017-08-04 2021-06-09 University of Florida Research Foundation, Incorporated Induction d'une immunité protectrice contre des antigènes
US11596677B2 (en) 2017-08-04 2023-03-07 University Of Florida Research Foundation, Incorporated Induction of protective immunity against antigens

Also Published As

Publication number Publication date
WO2009046449A1 (fr) 2009-04-09
US20110033501A1 (en) 2011-02-10
US8465755B2 (en) 2013-06-18
US20110256181A1 (en) 2011-10-20
US9040059B2 (en) 2015-05-26

Similar Documents

Publication Publication Date Title
US9040059B2 (en) Recombinant bacterium capable of eliciting a protective immune response against C. perfringens
Zekarias et al. Recombinant attenuated Salmonella enterica serovar Typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens
US9198950B2 (en) Recombinant bacterium comprising a toxin/antitoxin system
US11788056B2 (en) Induction of protective immunity against antigens
US6537558B2 (en) Methods of producing and using virulence attenuated poxR mutant bacteria
JP2002521345A (ja) 鳥類病原体を制御するため生きた弱毒化サルモネラワクチン
CA2526895A1 (fr) Attenuation regulee de vaccins vivants permettant d&#39;ameliorer l&#39;immunogenicite protectrice croisee
US20100303863A1 (en) Recombinant edwardsiella bacterium
US11766475B2 (en) Food safety vaccine to control Salmonella enterica and reduce Campylobacter in poultry
EP2526185A1 (fr) Bactérie renfermant un acide nucléique régulé par le rfah
KR102071743B1 (ko) 신규의 약독화된 시겔라 생백신
Choi et al. Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus)
Ding et al. Attenuated Listeria monocytogenes protecting zebrafish (Danio rerio) against Vibrio species challenge
US20230090746A1 (en) Attenuated salmonella synthesizing antigens for vaccinating against helicobacter pylori
CN111182920B (zh) 针对抗原的保护性免疫的诱导
Pedrosa Recombinant Bacillus subtilis spores as oral vaccines against aquaculture diseases
against Necrotic Recombinant Attenuated
Tinge NONRECOMBINANT AND RECOMBINANT AVIRULENT SALMONELLA LIVE VACCINES FOR POULTRY

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08836509

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 09.07.2010)

122 Ep: pct application non-entry in european phase

Ref document number: 08836509

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 12681711

Country of ref document: US